WO2017112792A1 - Tripartite modulators of endosomal g protein-coupled receptors - Google Patents

Tripartite modulators of endosomal g protein-coupled receptors Download PDF

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Publication number
WO2017112792A1
WO2017112792A1 PCT/US2016/068075 US2016068075W WO2017112792A1 WO 2017112792 A1 WO2017112792 A1 WO 2017112792A1 US 2016068075 W US2016068075 W US 2016068075W WO 2017112792 A1 WO2017112792 A1 WO 2017112792A1
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Prior art keywords
group
endosomal
nkir
compound
modulator
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PCT/US2016/068075
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English (en)
French (fr)
Inventor
Nigel W. Bunnett
Christopher J.H. Porter
Derek Cole
Gareth HICKS
Junya Shirai
Gavin Hirst
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Takeda Pharmaceutical Company Limited
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Priority claimed from AU2015905333A external-priority patent/AU2015905333A0/en
Priority to JP2018532435A priority Critical patent/JP7074673B2/ja
Priority to CN202210637197.4A priority patent/CN115531553A/zh
Priority to US16/064,061 priority patent/US11324832B2/en
Priority to AU2016377658A priority patent/AU2016377658B2/en
Priority to EA201891472A priority patent/EA201891472A1/ru
Priority to BR112018012425-7A priority patent/BR112018012425B1/pt
Priority to CN201680074910.1A priority patent/CN108712913B/zh
Application filed by Takeda Pharmaceutical Company Limited filed Critical Takeda Pharmaceutical Company Limited
Priority to CA3009325A priority patent/CA3009325A1/en
Priority to EP16831867.3A priority patent/EP3393519A1/en
Publication of WO2017112792A1 publication Critical patent/WO2017112792A1/en
Priority to US17/738,739 priority patent/US20230119819A1/en
Priority to JP2022078995A priority patent/JP7458681B2/ja
Priority to AU2022291659A priority patent/AU2022291659A1/en
Priority to JP2024041282A priority patent/JP2024073581A/ja

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Definitions

  • the present invention relates to tripartite compounds and their uses, and in particular to tripartite compounds that act as modulators to endocytosed G protein-coupled receptors.
  • G protein-coupled receptors are the largest family of cell-surface receptors (-850 members), participate in most pathophysiological processes, and are the target of -30% of therapeutic drugs (Audet, M. & Bouvier, M. Nat Chem Biol 2008, 4, 397-403).
  • Cell- surface GPCRs interact with extracellular ligands and couple to heterotrimeric G proteins, which trigger signaling pathways that originate from the plasma membrane, including formation of second messengers, transactivation of growth factor receptors, and regulation of ion channels.
  • Parrs recruit diverse signaling proteins to activated receptors at plasma and endosomal membranes and are essential mediators of signaling.
  • the MAPK cascades [ERK, c-Jun amino-terminal kinase (J K), p38] are the most thoroughly characterized Parrs-dependent signaling pathways.
  • the first evidence that Parrs are active participants in signaling was the observation that dominant negative mutants of Parr inhibited P2AR-induced activation of ERK1/2 (Daaka Y, et al. J Biol Chem 1998, 273, 685-688). Subsequently, Parrs were found to couple P2AR to c-Src and mediate ERK1/2 activation (Lutterall L. M. et al.
  • Parrs similarly participate in ERK1/2 signaling by other GPCRs, including neurokinin- 1 receptor (NKiR), protease-activated receptor 2 (PAR 2 ), angiotensin II type 1A receptor (ATIAR), and vasopressin V2 receptor (V2R).
  • NKIR neurokinin- 1 receptor
  • PAR 2 protease-activated receptor 2
  • ATIAR angiotensin II type 1A receptor
  • V2R vasopressin V2 receptor
  • modulating endocytosed GPCRs may advantageously provide a novel method of treating the vast number of diseases and disorders linked to this large family of receptors.
  • tripartite compounds are provided that are suitable for the modulation of endosomal GPCR signalling.
  • the tripartite compounds of the present invention can provide a novel method for the treatment and prevention of diseases and disorders mediated by these receptors. Accordingly, in one aspect the present invention provides a tripartite compound for targeting endosomal GPCR signaling of the formula (I): wherein
  • LA is a lipid anchor that promotes insertion of the compound into a plasma membrane;
  • L is a linker moiety of 1 nm to 50 nm in length; and
  • X is the modulator of the endosomal GPCR; or or a pharmaceutically acceptably salt thereof.
  • the present invention provides a tripartite compound for targeting endosomal neurokinin-1 receptor (NKiR) signaling of the formula (I): wherein
  • LA is a lipid anchor that promotes insertion of the compound into a plasma membrane;
  • L is a linker moiety of 1 nm to 50 nm in length, wherein L is represented by the formula
  • Z is the attachment group of the linker to the lipid anchor LA; wherein Z is defined by: al) Ci-Cioalkyl- -C 2 -Ci 0 alkenyl- -C 2 -C 10 alkynyl- -Ci-Ci 0 alkylC(O)-, -C 2 -
  • an optionally C-terminally amidated amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the amino acid is attached to the lipid anchor via its side-chain functional group; and
  • Y is the attachment group of the linker to the modulator X, which is a modulator of the endosomal neurokinin- 1 receptor (NKiR), wherein a2) Y is defined by a covalent bond, -0-, -NH-, -S- , -C(0)-, -C(0)NH- -C(0)0-
  • an alpha amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the alpha amino acid is optionally attached to the modulator of the endosomal NKiR via the side-chain functional group of at least one of said alpha amino acids; or
  • beta, gamma or delta amino acid comprising a side-chain functional group which is also found in aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein said beta, gamma or delta amino acid is optionally attached to the modulator of the endosomal NKiR via at least one of said side-chain functional groups; or c.
  • a peptide formed from alpha, beta, gamma or delta amino acids wherein the peptide comprises at least one alpha, beta, gamma or delta amino acid which has a side-chain functional group which is also found in aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; or combinations thereof, wherein said peptide is optionally attached to the modulator of the endosomal NKiR via at least one of said side-chain functional groups; m is 1 or 2; n is from 1 to 20; p is from 1 to 8; and
  • R 1 is defined by -F x -R 2 -F 2 -, wherein: - M is covalently linked to R 2 via F 1 (M-F ⁇ R 2 -), and
  • R 2 is further covalently linked to Y of the linker L via F 2 (M-F ⁇ R 2 -F 2 -Y-), and F 1 and F 2 independently from each other have a covalent bond to R 2 ;
  • R 2 is defined by: a3) covalent bond, b3) linear or branched C1-4 alkyl group, optionally having, as a substituent, a 5- to 7- membered aromatic or non-aromatic heterocyclic group, which optionally further contains in addition to carbon atoms 1 to 4 heteroatoms selected from the group consisting of oxygen atom, sulfur atom and nitrogen atom, and optionally further having one or two oxo as substituents, c3) 5- to 7-membered non-aromatic heterocyclic group, optionally further containing, besides carbon atoms, 1 or 2 nitrogen atoms, and optionally having 1 to 3 substituents selected from a group consisting of oxo, linear or branched C 1-6 alkyl, phenyl, linear or branched C 1-6 alkyl-carbonyl and C 1-6 alkyl-carbonylamino, d3) linear or branched C 1-6 alkyl group, optionally having substituent(s) selected from the group consisting of
  • Ci-6 alkoxy group a Ci-6 alkoxy group; e3) linear or branched C 1-6 alkoxy group, f3) C3-8 cycloalkyl group optionally having 1 or 2 substituents selected from a group consisting of a C 1-6 alkyl-carbonylamino group, a C 1-6 alkoxy-carbonylamino group and an amino group, g3) carbamoyl group, h3) linear or branched C 1-6 alkoxy-carbonyl group, i3) Ci-6 alkyl-carbamoyl group; or combinations thereof;
  • F 1 is defined by a covalent bond or by a moiety selected from the list of functional groups consisting of:
  • R 4 being hydrogen atom or linear or branched Ci to C 12 alkyl
  • R 2 is a hydrogen atom, linear or branched C 1-6 alkyl group, which optionally is halogenated;
  • R 3 and R are each independently a hydrogen atom or methyl, or R 3 and R 3 are optionally bonded to each other to form a ring together with a carbon atom bonded thereto;
  • the lipid anchor partitions into lipid membranes that are insoluble in non-ionic detergent at 4°C; or a pharmaceutically acceptable salt thereof.
  • LA is a lipid anchor, optionally promoting insertion of the compound into a plasma membrane, represented by formula (Ilaa), optionally by formula (Ila) or formula (Ilia):
  • R a is an optionally substituted C 1-12 alkyl, alkenyl, alkynyl, alkoxy group
  • the modulator of the endosomal NKiR is selected from at least one compound according to formula (VI) or formula (Vb)
  • R is methyl or cyclopropyl
  • R is a hydrogen atom or CH 3 ;
  • R 4 and R 5 are trifluoromethyl
  • R 6 is a hydrogen atom, methyl, ethyl or isopropyl
  • R is a hydrogen atom, methyl or chlorine atom
  • R is a hydrogen atom, a fluorine atom, a chlorine atom or methyl
  • the partial structure is or R 3 is hydrogen atom, and R 3 is hydrogen atom; or a pharmaceutically acceptable salt thereof.
  • the moiety -R 2 -F ⁇ M which is covalently linked to the linker L by -F 2 - as shown in the following structure, is as follows:
  • R 3 is hydrogen or -CH 3 ;
  • R 4 is hydro gen or fluorine; or a pharmaceutically acceptable salt thereof.
  • the moiety -R 2 -F ⁇ M which is covalently linked to the linker L by -F 2 - as shown in the following structure, is as follows:
  • the moiety -F 2 -R 2 -F ⁇ M which is covalently linked by Y to the linker L as shown in the following structures, is selected from the list of compounds consisting of: WO 2017/112792
  • R 3 is hydrogen atom or CH 3 ;
  • R 4 is hydrogen or fluorine or pharmaceutically acceptable salts thereof.
  • the moiety -F 2 -R 2 -F ⁇ M which is covalently linked by Y to the linker L as shown in the following structures, is selected from the list of compounds consisting of:
  • the compound is selected from the list of compounds consisting of the following structures:
  • R 3 is hydrogen atom or -CH 3 ; or a pharmaceutically acceptable salt thereof.
  • a prodrug of the tripartite compound is provided.
  • a pharmaceutical composition comprising the tripartite compound, or a prodrug thereof, is provided.
  • the pharmaceutical composition is for use as a tachykinin receptor antagonist.
  • the pharmaceutical composition is provided for the treatment of a disease or disorder mediated by endosomal NKiR signalling.
  • the pharmaceutical composition is provided for use in the prophylaxis or treatment of a lower urinary tract disease, a gastrointestinal disease or a central nervous system disease.
  • the pharmaceutical composition is provided for the prophylaxis or treatment of overactive bladder, irritable bowel syndrome, inflammatory bowel disease, vomiting, nausea, depression, anxiety neurosis, anxiety, generalised anxiety disorder (GAD), pelvic visceral pain or interstitial cystitis.
  • overactive bladder irritable bowel syndrome
  • inflammatory bowel disease vomiting, nausea, depression, anxiety neurosis, anxiety, generalised anxiety disorder (GAD), pelvic visceral pain or interstitial cystitis.
  • GAD generalised anxiety disorder
  • the pharmaceutical composition is provided for treatment of a disease or disorder mediated by endosomal NKiR signaling selected from the list of chemotherapy -induced nausea and vomiting (CINV), cyclic vomiting syndrome, postoperative nausea and vomiting, affective and addictive disorders including depression and anxiety, generalised anxiety disorder (GAD), gastrointestinal disorders including inflammatory bowel disease, irritable bowel syndrome, gastroparesis and functional dyspepsia, chronic inflammatory disorders including arthritis, respiratory disorders including COPD and asthma, urogenital disorders, sensory disorders and pain including somatic pain and visceral pain, pruritus, viral and bacterial infections and proliferative disorders (cancer), and combinations thereof.
  • CINV chemotherapy -induced nausea and vomiting
  • GAD generalised anxiety disorder
  • gastrointestinal disorders including inflammatory bowel disease, irritable bowel syndrome, gastroparesis and functional dyspepsia
  • chronic inflammatory disorders including arthritis
  • respiratory disorders including COPD and asthma
  • urogenital disorders urogenital disorders
  • sensory disorders and pain including somatic pain and
  • the disease or disorder mediated by endosomal NKiR signaling is somatic pain or visceral pain.
  • the disease or disorder mediated by endosomal NKiR signaling is a chronic disease or disorder.
  • the tripartite compound is used in the treatment of a disease or disorder mediated by endosomal NKiR signalling.
  • a pharmaceutical composition comprising a therapeutically effective amount of a tripartite compound, or pharmaceutically acceptable salts thereof, together with at least one pharmaceutically acceptable carrier or diluent.
  • a tripartite compound for targeting endosomal GPCR signaling of the following formula is provided:
  • the present invention provides a tripartite compound for targeting endosomal GPCR signaling of the formula (Ie): wherein
  • LA is a lipid anchor that promotes insertion of the compound into a plasma membrane
  • L is a linker moiety of 1 nm to 50 nm in length
  • X is a modulator of an endosomal GPCR; wherein, the lipid anchor partitions into lipid membranes that are insoluble in non-ionic detergent at 4°C; or pharmaceutically acceptable salts thereof.
  • the present invention provides a tripartite compound for targeting endosomal GPCR signaling of the formula (la): wherein
  • LA is a lipid anchor that promotes insertion of the compound into a plasma membrane represented by formulae (Ila) or (Ilia):
  • R la is an optionally substituted C 1-12 alkyl group
  • R 2a and R a are independently H or C 1-3 alkyl
  • R 4a is C, 0, NH or S
  • L is a linker group of 1 nm to 50 nm in length
  • X is a modulator of an endosomal GPCR; or pharmaceutically acceptable salts thereof.
  • the present invention provides a tripartite compound for targeting endosomal GPCR signaling of the formula (lb):
  • LA is a lipid anchor that promotes insertion of the compound into a plasma membrane represented by formulae (Ila) or (Ilia):
  • R a is an optionally substituted C 1-12 alkyl group
  • R a and R a are independently H or C 1-3 alkyl
  • R 4a is C, (X NH or S: represents a single or double bond;
  • L is represented by the formula (IV a):
  • Z is the attachment group between the linker and the lipid anchor and is -Ci-Cioalkyl-
  • Z together with the adjacent amine, is an optionally C-terminal ami dated amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the amino acid is attached to the lipid anchor via its side-chain functional group;
  • Y is the attachment group between the linker and the modulator of an endosomal GPCR and is -0-, -NH-, -S- , -C(0)-, -C(0)NH- -C(0)0- or -C(0)S-; or
  • Y together with the adjacent ami do group, is an amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the amino acid is attached to the modulator of an endosomal GPCR via its side-chain functional group; m is 1 or 2; n is from 1 to 20; p is from 1 to 8; and
  • X is a modulator of an endosomal GPCR; or pharmaceutically acceptable salts thereof.
  • the present invention provides a method of modulating endosomal GPCR signaling comprising contacting an endosomal GCPR with a tripartite compound of formula (Ie) as herein defined.
  • the present invention provides a method of modulating endosomal GPCR signaling in a subject in need thereof comprising administering to the subject an effective amount of a tripartite compound of formula (Ie) as herein defined.
  • the present invention provides a method for the treatment of a disease or disorder mediated by endosomal substance P (SP) or neurokinin 1 receptor (NKiR) signaling comprising administering to a subject in need thereof an effective amount of a tripartite compound of formula (Ie) as herein defined.
  • SP endosomal substance P
  • NKIR neurokinin 1 receptor
  • the present invention provides a method for the treatment of a disease or disorder mediated by endosomal CGRP signaling comprising administering to a subject in need thereof an effective amount of a compound of formula (Ie) as herein defined.
  • Figure 1 Schematic representation of the tripartite compound of the present invention illustrating its different moieties and the linkage pattern between the moieties.
  • Figure 2 Synthetic procedure for preparing the tripartite compound Compound A.
  • Figure 3 Synthetic procedure for preparing the tripartite compound Compound B.
  • Figure 4 Synthetic procedure for preparing the core modulator moieties Compound C-l and Compound C-2, before coupling these modulators via -Y- to the linker.
  • Figure 5 Graphical representations of NKiR subcellular trafficking.
  • A BRET analysis of the proximity between NKiR-RLuc8, -arrl/2-YFP, KRas-Venus or Rab5a-Venus.
  • B-C SP-induced BRET in HEK293 cells between NKiR-RLuc8 and ⁇ -arrl-YFP (B) or -arr2- YFP (C).
  • D-F SP-induced BRET in HEK293 cells between NKiR-RLuc8 and KRas- Venus (D, E) or Rab5a-Venus (D, F).
  • E, F show effects of endocytic inhibitors.
  • FIG. 6 Graphical representations of NKiR subcellular signaling
  • A, C, E FRET biosensors for detection of activated ERK in the nucleus (NucEKAR), activated PKC in the cytosol (CytoCKAR), and cAMP in the cytosol (CytoEpac2).
  • B, D, E, G, H show area under curve (AUC).
  • FIG. 7 Graphical representations of G-protein-dependent NKiR signaling.
  • A-D SP- induced BRET in HEK293 cells between NKiR-RLuc8 and KRas-Venus (A), NKiR- RLuc8 and Rab5a-Venus (B), Ga q -RLuc8 and Gy2-Venus (C), and Go q -RLuc8 and Rab5a- Venus (D) after 15 min.
  • E-G SP- induced BRET in HEK293 cells between NKiR-RLuc8 and KRas-Venus (A), NKiR- RLuc8 and Rab5a-Venus (B), Ga q -RLuc8 and Gy2-Venus (C), and Go q -RLuc8 and Rab5a- Venus (D) after 15 min.
  • Figure 8 Graphical representations of SP-evoked NKiR endocytosis and excitability in spinal neurons.
  • B-D Effects of endocytic inhibitors on SP-evoked firing of rat spinal neurons. B shows representative traces. C shows firing rate normalized to response at 2 min (P>0.05). D shows firing time as duration of response to last action potential.
  • n 7 cells per treatment, 17 rats, *P ⁇ 0.05, **P ⁇ 0.01.
  • Figure 9 Graphical representations of of NKiR endocytosis and ERK signaling in spinal neurons.
  • Vehicle (Veh), Dy4, Dy4 inact, PS2 or PS2 inact was injected intrathecally (i.t.) to rats 30 min before intraplantar (i.pl.) injection of vehicle or capsaicin (Cap).
  • A shows ratio of cytosolic to plasma membrane NKiR-IR
  • B shows ratio of pERK-IR to NeuN- IR neurons.
  • LI, LII lamina I, II. Scale A, 10 ⁇ ; C, 25 ⁇ . **P ⁇ 0.01, ***P ⁇ 0.001.
  • n 3 rats; NKiR, analysis of 6 cells per condition, p-ERK analysis of 6 fields per condition.
  • Figure 10 Graphical representations of mechanical hyperalgesia, inflammatory edema and motor coordination in mice.
  • A-D Vehicle (Veh) or endocytic inhibitor was injected intrathecally 30 min before intraplantar injection of capsaicin (Cap) or vehicle into the left paw.
  • A, B show von Frey withdrawal responses of injected left paw
  • C shows edema responses of injected left paw.
  • D Vehicle or endocytic inhibitor was injected intrathecally 30 min before measurement of rotarod latency.
  • E-H siRNA to dynamin-1 (E, F), arrl+2 (G, H) or control siRNA was injected intrathecally 24-48 h before intraplantar injection of capsaicin into the left paw.
  • E, G show knockdown in the dorsal horn of dynamin-1 determined by Western blot (E), and arr 1+2 determined by RT-PCR (G).
  • F, H show von Frey withdrawal responses of injected left paw.
  • I Vehicle or U0126 was injected intrathecally 30 min before intraplantar injection of capsaicin or vehicle into the left paw. von Frey withdrawal responses of injected left paw were measured.
  • L
  • CFA was injected into the left paw 36 h before intrathecal injection of vehicle or endocytic inhibitor, von Frey withdrawal responses of injected left paw were measured.
  • FIG. 11 Graphical representations of targeting endosomal NKiR using tripartite probes.
  • A Structure of tripartite probes with Cy5, spantide or L-733,060 cargo.
  • B Uptake of Cy5- ethyl ester or Cy5-cholestanol (Cy5-Chol) (30 min, 37°C) in HEK293 cells. Asterisk, extracellular; arrowheads, plasma membrane; arrows, endosomes. Scale, 10 ⁇ .
  • C SP activation of nuclear ERK in HEK293 cells.
  • Pre-incubation Cells were pre-incubated with spantide (Span), Compound 1 or SR140,333 (SR) for 30 min, and then immediately stimulated with SP (in the presence of inhibitors).
  • Figure 12 Graphical representations of Targeting of endosomal NKiR in spinal neurons using tripartite probes.
  • A-C SP-evoked firing of rat spinal neurons.
  • A shows representative traces.
  • B shows firing rate normalized to response at 2 min (events.2 min "1 : Compound 1, 196.6 ⁇ 81.6; spantide, 242.6 ⁇ 95.9; P>0.05).
  • C shows firing time as duration of response to last action potential.
  • n 7 cells per treatment, 18 rats, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • D E.
  • CFA was injected into the left paw of mice 36 h before intrathecal injection of vehicle, SR140,333 (SR), spantide or Compound 1.
  • von Frey withdrawal responses of injected left paw were measured in different groups of mice 36-39 h (H) or 39-42 h (I) after CFA.
  • J. Vehicle (Veh), L-733,060 (L733) or Compound 4 was injected intrathecally to mice 3 h before intraplantar injection of capsaicin into the left paw. von Frey withdrawal responses of injected left paw are shown. **P ⁇ 0.01, ****P ⁇ 0.0001 to Cy5-cholestanol or vehicle, (n), mouse number.
  • FIG. 13 Graphical representations of CGRP-mediated mechanical hyperalgesia in mice.
  • A B. Vehicle (Veh), Olcegepant (Olceg), Compound 4 or free CGRP 8"37 was injected intrathecally 3 h before intraplantar injection of capsaicin (Cap) into the left paw.
  • A shows von Frey withdrawal responses of the injected left paw
  • B shows von Frey withdrawal responses of the non-injected right paw.
  • C D. Vehicle, Olcegepant, Compound 4 or free CGRP 8"37 was injected intrathecally 3 h before intraplantar injection of formalin into the left paw. Nocifensive behavior was measured for 60 min.
  • C shows response kinetics
  • D shows responses during first and second phases.
  • E E.
  • CFA Complete Freund's adjuvant
  • endosomal GPCRs convey sustained signals that underlie excitation and nociceptive transmission in spinal neurons.
  • GPCR endothelin type B (ET B R) localised in caveolae may provide an effective anti-tumour therapy (see WO2005/097199).
  • the ET B R has been shown to mediate diverse physiological effects on vasoconstriction, cellular development, differentiation and mitogenesis (Yamaguch, T. et al. Eur. J. Biochem 2003, 270, 1816-1827). Distinct from endosomes, caveolae are small flask-shaped or cup- shaped pits formed in the cell membrane and unlike endosomes, caveolae are not coated with clathrin.
  • a tripartite compound is provided according to formula (I) for modulating endosomal GPCR signaling.
  • the present invention provides a method of modulating endosomal GPCR signaling comprising administering to a subject in need thereof an effective amount of a compound of formula (Ie) as herein defined.
  • trimer compound refers to compounds comprising an endosomal GPCR modulator covalently bound to a linker group, the linker group being covalently bound to a lipid anchor capable of anchoring the compound of formula (I) or (Ie) to the lipid bilayer of a cell membrane and ultimately, to the membrane of an early endosome.
  • lipid anchor denotes moieties that are capable of partitioning into lipid membranes and thereby anchoring the compound of formula (I) into the lipid membrane.
  • the partition into the lipid membrane may occur directly from the extracellular or vesicular luminal space or may occur laterally from the lipid bilayer.
  • the lipid anchor may be characterized by its ability to partition into lipid membranes whereby said lipid membranes are characterized by insolubility in non-ionic detergents at 4°C.
  • lipid anchors include, but are not limited to cholesterol, cholestanol, sphingolipid, GPI-anchor or saturated fatty acid derivatives. Many such lipid anchors have been described in the art, for example, in WO2005/097199, the entirety of which is incorporated herein by reference.
  • the li id anchor is a moiety selected from formulae (Ila) or (Ilia):
  • R a is an optionally substituted C 1-12 alkyl group;
  • R 2a and R a are independently H or Ci -3 alkyl;
  • R 4a is -CH 2 -, -0-, -NH-, -S-, -NH(CH 2 )aOP0 3 " -, -NH(CH 2 ) a S0 2 CF 2 - -NH(CH 2 ) a S0 2 NH- -NHCONH-, -NHC(0)0-, -NHCH(CONH 2 )(CH 2 ) b C(0)0- -NHCH(COOH)(CH 2 ) b C(0)0- -NHCH(CONH 2 )(CH 2 ) b CONH- -NHCH(COOH)(CH 2 ) b CONH-, -NHCH(CONH)(CH 2 ) 4 NH((CO)CH 2 0) e - or -NHCH(COOH)(CH 2 ) NH((CO)CH 2 0) e -; a is an integer from 2 to 3; b is an integer from 1 to 2; e is an integer from 0 to
  • the lipid anchor is a moiety selected from formulae (Via), (Vila), (Villa) or (IXa):
  • R 4a is as described above; represents a single or double bond; represents a single, double or triple bond; each occurrence of R 3a is independently -NH-, -0-, -S-, -0C(0)-, -NHC(O)-, -NHCONH-, -NHC(0)0- or -NHS(0 2 )-; each occurrence of R a is independently a C 14-30 alkyl group optionally substituted by fluorine, optionally 1 to 4 fluorine atoms; each occurrence of R 7a is independently NH 2 , NHCH 3 , OH, H, halo gen or O, provided that when R 7a is NH 2 , NHCH 3 , OH, H or halogen then is a single bond and when R 7a is
  • each occurrence of R a is independently H, OH or is absent when represents a triple bond;
  • R a is a C 10-30 alkyl group optionally substituted by fluorine, optionally 1 to 4 fluorine atoms; and each occurrence of R 10a is independently a C24-40 alkylene group, a C24-40 alkenylene group or a C24-40 alkynylene group optionally substituted by fluorine, optionally 1 to 4 fluorine atoms.
  • the lipid anchor is a moiety selected from formulae (Xa) or (XIa):
  • R 1 a represents a single or double bond; represents a single, double or triple bond; each occurrence of R 1 a is independently -O- or -CO(CH 2 ) a (CO) b O-, wherein a is an integer from 1 to 3 and b is an integer from 0 to 1 ;
  • R 14a is -O- or -OC(O) -; each occurrence of R 15a is independently selected from a C 16-30 alkyl group optionally substituted with fluorine, optionally 1 to 4 fluorine atoms;
  • R 16a is -PO3 CH2-, -SO3CH2-, -CH 2 - -CO2CH2- or a direct bond;
  • R 17a is -NH-, -0-, -S-, -OC(O)-, -NHC(O)-, -NHCONH-, -NHC(0)0- or -NHS(0 2 )-;
  • R 18a is NH 2 , NHCH 3 , OH, H, halogen or O;
  • R 19a is a C 16-30 alkyl group optionally substituted with fluorine, optionally 1 to 4 fluorine atoms; and each R a is a C(0)Ci3-25alkyl group optionally substituted with a group of the following formulae:
  • R zza is -NH-, -0-, -S-, -OC(O)-, -NHC(O)-, -NHCONH-, -NHC(0)0- or -NHS(0 2 )
  • R Zia is -O- or -OC(O) -; each occurrence of R 24a is independently selected from a C 16-30 alkyl group optionally substituted with fluorine, optionally 1 to 4 fluorine atoms;
  • R 25a is -CO(CH 2 )a(CO) b O- or -CO(CH 2 ) a (CO) b NH-, wherein a is an integer from 1 to 3 and b is an integer from 0 to 1;
  • R 26a is a C4-20 alkyl group optionally substituted with fluorine, optionally 1 to 4 fluorine atoms.
  • the lipid anchor is a moiety selected from formulae (Xlla), (Xllla), XlVa) or (XVa):
  • each occurrence of R a is independently selected from -NH-, -0-, -NH(CH 2 ) c OP0 3 -, -NH(CH 2 ) c S0 2 NH- -NHCONH-, -NHC(0)0- -CO(CH 2 ) b (CO) a NH- -CO(CH 2 ) b (CO) a O- -CO(CH 2 ) b S-, -CO(CH 2 ) b OP0 3 - -CO(CH 2 ) b S0 2 NH-, -CO(CH 2 ) b NHCONH- -CO(CH 2 ) b OCONH-, -CO(CH 2 ) b OS0 3 -, or
  • linker as herein used relates to the part of the compound that links the modulator of an endosomal GPCR to the lipid anchor. It will be understood that the linker should be selected such that it does not compete with the modulator of an endosomal GPCR at the ligand binding site. Nor should the linker partition into the lipid membrane.
  • the linker group should optionally be of a length of between 1 nm to 50 nm in order to allow the modulator of an endosomal GPCR to interact with the receptor when the lipid anchor is anchored in the endosome membrane.
  • the linker group will comprise one or more polyethelene glycol units.
  • the linker, or subunits of the linker may be amino acid residues, derivatised or functionalised amino acid residues, polyethers, ureas, carbamates, sulphonamides or other subunits that provide adequate distance between the modulator of an endosomal GPCR and the lipid anchor without interfering in the function of either group.
  • the linker is represented by a moiety of the formula (IV a):
  • Z is the attachment group of the linker to the lipid anchor and is -Ci-Cioalkyl- -C 2 -Cioalkenyl- -C 2 -Ci 0 alkynyl-, -C C 10 alkylC(OK -C 2 -Ci 0 alkenylC(O)- or
  • Z together with the adjacent amine, is an optionally C-terminal ami dated amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the amino acid is attached to the lipid anchor via its side-chain functional group;
  • Y is the attachment group between the linker and the modulator of an endosomal GPCR and is -0-, -NH-, -S- , -C(O)-, -C(0)NH- -C(0)0- or -C(0)S-; or
  • Y together with the adjacent ami do group is an amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the amino acid is attached to the modulator of an endosomal GPCR via its side-chain functional group;
  • Y is the attachment group of the linker to the modulator X, which is a modulator of the endosomal neurokinin- 1 receptor (NKiR), wherein a2)
  • Y is defined by -0-, -NH-, -S- , -C(0)-, -C(0)NH- -C(0)0- -O-C(O)-, -NH-
  • an alpha amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the alpha amino acid is optionally attached to the modulator of the endosomal NKiR via the side-chain functional group of at least one of said alpha amino acids; or
  • beta, gamma or delta amino acid comprising a side-chain functional group which is also found in aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein said beta, gamma or delta amino acid is optionally attached to the modulator of the endosomal NKiR via at least one of said side-chain functional groups; or c.
  • a peptide formed from alpha, beta, gamma or delta amino acids wherein the peptide comprises at least one alpha, beta, gamma or delta amino acid which has a side-chain functional group which is also found in aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; or combinations thereof, wherein said peptide is optionally attached to the modulator of the endosomal NKiR via at least one of said side-chain functional groups; m is 1 or 2; n is from 1 to 20; and p is from 1 to 8.
  • the linker is represented by a moiety of the formula (XXa): wherein each occurrence of R l la is independently any side chain of a naturally occurring, derivatised or functionalised amino acid residue: m is an integer from 3 to 80; and n is an integer from 0 to 1.
  • the linker is represented by a moiety of the formula (XXIa):
  • XXIa wherein m is an integer from 0 to 40; n is an integer from 0 to 1 ; each occurrence of o is independently an integer from 1 to 5; each occurrence of R l la is independently any side chain of a naturally occurring, derivatised or functionalised amino acid residue; and wherein the SO2 terminus is bound to the lipid anchor and the N-terminus is bound to the modulator of an endosomal GPCR.
  • the linker is represented by a moiety of the formula (XXIIa):
  • XXIIa wherein m is an integer from 0 to 40; n is an integer from 0 to 1 ; each occurrence of o is independently an integer from 1 to 5; each R a is independently NH or O; each occurrence of R l la is independently any side chain of a naturally occurring, derivatised or functionalised amino acid residue: and wherein the C(0)-terminus is bound to the lipid anchor and the R a -terminus is bound to the modulator of an endosomal GPCR.
  • linker moieties have been described in WO2005/097199, the entirety of which is incorporated herein by reference.
  • modulator of an endosomal GPCR refers to agonists and antagonists or inhibitors of G protein-coupled receptors that are endocytosed into endosomes.
  • the GPCR modulator will be an agonist of the receptor. In another embodiment, the GPCR modulator will be an antagonist or inhibitor of the receptor.
  • the GPCR modulator may be in any form including, but not limited to, an organic molecule, a polypeptide sequence, a hormone, a protein fragment or a derivative of any of these.
  • the GPCR modulator optionally is an organic molecule, or, alternatively or additionally, a hormone, or a derivative of these.
  • endosomal GPCR signaling refers to the signal transduced by an activated G protein-coupled receptor that has been endocytosed into an endosome, optionally an early endosome.
  • endosomal GPCR signaling will be signaling that is first transduced at the plasma membrane and is maintained when the receptor is endocytosed into early endosomes.
  • the endosomal GPCR signaling will be signaling that requires receptor endocytosis and/or occurs exclusively on endosomal membranes, for example, ⁇ - arrestin mediated signaling. It is believed that Parrs interact with agonist-occupied, G protein-coupled receptor kinase (GRK)-phosphorylated GPCRs at the cell surface and promote the transfer of ligand-bound receptor from the cell surface to early endosomes via dynamin- and clathrin-dependent endocytosis. It has recently been discovered that this pathway can mediate a second series of endosomal GPCR signaling that is distinct from G protein-dependent signaling at the plasma membrane.
  • GRK G protein-coupled receptor kinase
  • Class C GPCRs e.g., bradykinin B 2 receptor
  • Parrs internalize with Parrs into endosomes followed by rapid dissociation of Parr upon agonist removal. It is believed that the extent of Parr-induced MAPK signaling depends on the affinity of the receptor for Parrs, which depends on the receptor structure and on which of the seven mammalian GRKs phosphorylate the receptor. Thus, activation of ATIAR and V 2 R causes greater phosphorylation of Parr-bound ERK1/2 than activation of oci b AR and P 2 AR, suggesting that the class B receptors signal more robustly through this pathway.
  • Class B GPCRs include the secretin receptor, VPACi receptor, VPAC 2 receptor, PACi receptor, glucagon receptor, growth hormone releasing hormone (GHRH) receptor, glucagon-related peptide 1 (GLP-1) receptor, glucagon-related peptide 2 (GLP-2) receptor, gastric inhibitory polypeptide (GIP) receptor, corticotropin releasing factor 1 (CRF1) receptor, corticotropin releasing factor 2 (CRF2) receptor, parathyroid hormone 1 (PTH1) receptor, parathyroid hormone 2 (PTH2) receptor, calcitonin receptor-like receptor, calcitonin receptor, angiotensin II receptor type 1, vasopressin receptor 2, calcitonin gene related peptide (CGRP) receptor, neurokinin 1 receptor (NKiR), and protease activated-2 receptor (PAR 2 ).
  • GHRH growth hormone releasing hormone
  • GLP-1 glucagon-related peptide 1
  • GLP-2 glucagon-related peptide
  • LA is a lipid anchor selected from cholesterol, cholestanol, sphingolipid, a GPI- anchor or a saturated fatty acid derivative.
  • LA is a lipid anchor selected from moieties of formulae (Ila), (Ilia), (Vila), (Villa), (IXa), (Xa), (XIa), (Xlla), (XHIa) and (IXa).
  • LA is a lipid anchor selected from moieties of formulae (Ila) or (Ilia).
  • L is a linker moiety comprising one or more subunits, the subunits comprising polyethelene glycol units, amino acid residues, derivatised or functionalised amino acid residues, polyethers, ureas, carbamates and/or sulphonamides.
  • L is a linker moiety represented by formulae (IV a), (XXa), (XXIa) or (XXIIa).
  • f) L is a linker moiety represented by formula (IV a).
  • X is an agonist of an endosomal GPCR, optionally of endosomal neurokinin- 1 receptor.
  • X is an antagonist of an endosomal GPCR, optionally of endosomal neurokinin- 1 receptor.
  • LA is a lipid anchor represented by formulae (Ila) or (Ilia).
  • the present invention provides tripartite compounds of the formula (I) represented by formula (la): wherein
  • LA is a lipid anchor that promotes insertion of the compound into a plasma membrane represented by formulae (Ila) or (Ilia):
  • R la is an optionally substituted C 1-12 alkyl group
  • R 2a and R a are independently H or C 1-3 alkyl
  • R 4a is C, O, NH or S; and represents a single or double bond
  • L is a linker group of 1 nm to 50 nm in length
  • X is a modulator of an endosomal GPCR, optionally endosomal neurokinin- 1 receptor; or pharmaceutically acceptable salts thereof.
  • LA is a lipid anchor represented by formulae (Ila) or (Ilia) and L is a linker represented by formula (IV a).
  • the present invention provides tripartite compounds of the formula (I) represented by formula (lb):
  • LA is a lipid anchor that promotes insertion of the compound into a plasma membrane represented by formulae (Ila) or (Ilia):
  • R la is an optionally substituted C 1-12 alkyl group
  • R 2a and R a are independently H or C 1-3 alkyl
  • R 4a is C, 0, NH or S; represents a single or double bond
  • L is a linker represented by the formula (IV a):
  • Z is the attachment group between the linker and the lipid anchor and is-Ci-Cioalkyl-,
  • Z together with the adjacent amine, is an optionally C-terminal ami dated amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the amino acid is attached to the lipid anchor via its side-chain functional group;
  • Y is the attachment group between the linker and the modulator of an endosomal GPCR and is -0-, -NH-, -S- , -C(0)-, -C(0)NH- -C(0)0- or -C(0)S-; or
  • Y together with the adjacent ami do group is an amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the amino acid is attached to the modulator of an endosomal GPCR via its side-chain functional group; or
  • Z is the attachment group of the linker to the lipid anchor LA; wherein Z is defined by: al) Ci-Cioalkyl- -C 2 -Ci 0 alkenyl- -C 2 -Ci 0 alkynyl- -Ci-Ci 0 alkylC(O)-, -C 2 - CioalkenylC(O)- or -C 2 -CioalkynylC(0)-; or
  • an optionally C-terminally amidated amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the amino acid is attached to the lipid anchor via its side-chain functional group; and
  • Y is the attachment group of the linker to the modulator X, which is a modulator of the endosomal neurokinin- 1 receptor (NKiR), wherein a2) Y is defined by -0-, -NH-, -S- , -C(0)-, -C(0)NH- -C(0)0- -O-C(O)-, -NH-
  • an alpha amino acid selected from aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein the alpha amino acid is optionally attached to the modulator of the endosomal NKiR via the side-chain functional group of at least one of said alpha amino acids; or
  • beta, gamma or delta amino acid comprising a side-chain functional group which is also found in aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; wherein said beta, gamma or delta amino acid is optionally attached to the modulator of the endosomal NKiR via at least one of said side-chain functional groups; or c.
  • a peptide formed from alpha, beta, gamma or delta amino acids wherein the peptide comprises at least one alpha, beta, gamma or delta amino acid which has a side-chain functional group which is also found in aspartic acid, glutamic acid, asparagine, glutamine, histidine, cysteine, lysine, arginine, serine or threonine; or combinations thereof, wherein said peptide is optionally attached to the modulator of the endosomal NKiR via at least one of said side-chain functional groups; m is 1 or 2; n is from 1 to 20; p is from 1 to 8; and X is a modulator of an endosomal GPCR; or pharmaceutically acceptable salts thereof. In a preferred embodiment, X is a modulator of the endosomal NKi receptor.
  • the NKiR rapidly and completely redistributes to endosomes at sites of pain transmission in the spinal cord (Marvizon, J. C. et al. JNeurosci 1997, 17, 8129-8136) and inflammation in the vasculature (Bowden, J. J. et al. Proc Natl Acad Sci USA 1994, 91 , 8964-8968), and is believed to internalize in patients with chronic pain and inflammation (Jarcho, J. M. et al. Pain, 2013).
  • NKiR signaling generates subcellular signals that underlie neuronal activation and hyperalgesia.
  • Painful and pro-inflammatory stimuli including the transient receptor potential vanilloid 1 agonist capsaicin, stimulate SP release from primary sensory nociceptors in laminae I, II of the dorsal horn, where SP stimulates NKiR endocytosis in spinal neurons.
  • the NKiR antagonist spantide was unable to ameliorate capsaicin-evoked hyperalgesia, probably due to metabolism and clearance of the compound.
  • NKiR antagonist when the same NKiR antagonist was incorporated into the tripartite compounds of the present invention, hyperalgesia was inhibited for more than 120 minutes, suggesting that antagonism of the NKiR in endosomes is an effective treatment against pain.
  • NKiR activation has been implicated in the treatment and prevention of a wide variety of disorders including depression and mood disorders, anxiety disorders, substance addiction-related disorders, alcohol-related disorders, sleep disorders, eating disorders, autism spectrum disorders, attention-deficit/hyperactivity disorder personality disorders, and cancer. Modulators of NKiR may also be useful for the treatment and prevention of inflammation, allergic disorders, neurological disorders, emesis, pain and cancer.
  • the present invention provides tripartite compounds of the formula (I) represented by formula (Ic): wherein
  • LA is a lipid anchor that promotes insertion of the compound into a plasma membrane
  • L is a linker moiety of 1 nm to 50 nm in length
  • X is a modulator of the endosomal NKiR; wherein, in a preferred embodiment of the present invention, the lipid anchor partitions into lipid membranes that are insoluble in non-ionic detergent at 4°C; or a pharmaceutically acceptable salt thereof.
  • the compound of formula (Ic) is selected from:
  • the individual moieties of the tripartite compound of the present invention including X. L, LA, Y, Z, the spacer between Y and Z, M, F 1 , F 2 , R 1 and R 2 which are linked to each other as schematically described in Figure 1.
  • the modulator X is formed from the moieties M, and R 1 , wherein R 1 includes the moieties F 1 , R 2 and F 2 .
  • the part F 1 is used to covalently link M with R 2
  • F 2 is used to covalently link R 2 with Y, Y being a part of the linker L.
  • Moieties F 1 and F 2 are covalently linked to R 2 without being limited to any specific substitution pattern.
  • the bonding pattern of F 1 and F 2 at R 2 is only limited by the availability of the functional groups present on F 1 , F 2 and R 2 .
  • Preferred modulator moieties are as indicated by the following structures: Moiety -R 2 -F ⁇ M, which is covalently linked to the linker L by -F 2 - as shown in the following structure. Moiety -R 2 -F x -M under this definition does not include F 2 which is only indicated in the structure for further illustration:
  • R 3 is hydrogen or -CH 3 ;
  • R 4 is hydro gen or fluorine; or a pharmaceutically acceptable salt thereof.
  • Moiety -R 2 -F ⁇ M which is covalently linked to the linker L by -F 2 - as shown in the following structure.
  • Moiety -R 2 -F x -M under this definition does not include F 2 which is only indicated in the structure for further illustration:
  • R 3 is hydrogen atom or CH 3 ;
  • R 4 is hydrogen atom or fluorine; or pharmaceutically acceptable salts thereof.
  • moieties -F 2 -R 2 -F ⁇ M which are covalently linked by Y to the linker L as shown in the following structures (Moieties -F 2 -R 2 -F x -M under this definition do not include Y which is only indicated in the three structures for further illustration:
  • tripartite compounds X-L-LA having the following structures:
  • R 3 is hydrogen atom or -CH 3 ; or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method for the treatment of a disease or disorder mediated by endosomal NKiR signaling, comprising administering to a subject in need thereof an effective amount of a compound of formula (Ic) as herein defined.
  • the present invention provides the use of a compound of formula (Ic) as herein defined in the manufacture of a medicament for the treatment of a disease or disorder mediated by endosomal NKiR signaling.
  • the present invention provides the use of the tripartite compounds for the treatment of a disease or disorder mediated by endosomal NKiR signalling.
  • the present invention provides a compound of formula (Ic) as herein defined for use in the treatment of a disease or disorder mediated by endosomal NKiR signaling.
  • the disease or disorder mediated by endosomal NKiR signaling is selected from chemotherapy -induced nausea and vomiting (CINV), cyclic vomiting syndrome, postoperative nausea and vomiting, affective and addictive disorders including depression and anxiety, generalised anxiety disorder (GAD), gastrointestinal disorders including inflammatory bowel disease, irritable bowel syndrome, gastroparesis and functional dyspepsia, respiratory disorders including COPD and asthma, urogenital disorders, sensory disorders and pain including somatic pain and visceral pain, pruritus, viral and bacterial infections and proliferative disorders (cancer).
  • CINV chemotherapy -induced nausea and vomiting
  • GAD generalised anxiety disorder
  • gastrointestinal disorders including inflammatory bowel disease, irritable bowel syndrome, gastroparesis and functional dyspepsia
  • respiratory disorders including COPD and asthma
  • urogenital disorders urogenital disorders
  • sensory disorders and pain including somatic pain and visceral pain, pruritus, viral and bacterial infections and proliferative disorders (cancer).
  • pain includes chronic
  • neuropathic pain syndromes including diabetic neuropathy, chemoterapeutically induced neuropathic pain, sciatica, non-specific lower back
  • the modulator of an endosomal GPCR is an inhibitor of the endosomal CGRP receptor.
  • CGRP neuropeptide calcitonin gene related peptide
  • CGRP CGRP receptor
  • CLR calcitonin receptor-like receptor
  • RAMP1 receptor activity modifying protein- 1
  • CGRP binding and receptor activation has been shown to lead to phosphorylation of CLR and internalization of the CGRP-R as a stable complex (Hilairet, S. et al., J. Biol. Chem., 2001, 276, 42182-42190). Internalization was found to be both dynamin- and ⁇ -arrestin dependent, indicating that the formation of a trinary complex between CLR, RAMP1 and ⁇ -arrestin leads to chathin-coated pit mediated endocytosis.
  • Telcagepant Early studies with the selective CGRP-R antagonist Telcagepant showed promising results with administration of the compound resulting in the effective treatment of moderate to severe migraine attacks with a primary endpoint of pain relief at 2 hours (Ho, T. W. et al., Neurology, 2008, 70(16) 1304-1312). Telcagepant also showed promise as an effective treatment for sustained pain relief over 24 hours and relief from migraine related symptoms such as photophobia, phonophobia and nausea. However, a Phase Ila clinical trial of the compound was ceased as Telcagepant was found to significantly increase levels of the hepatic liver enzyme alanine transaminase to an unacceptable level for the patient.
  • CGRPg-37 Another antagonist identified for the CGRP-R is CGRPg-37.
  • CGRP exists in two forms in humans, a-CGRP and ⁇ -CGRP. These forms are derived from separate genes and differ with respect to three amino acids, but exhibit similar biological functions (Durham, P. L and Vause, C. V. CNS Drugs, 2010, 24(7), 539-548). Binding studies of the 37-amino acid neuropeptide a-CGRP have revealed that the first seven N-terminal amino acids are essential for receptor activation. SAR studies have demonstrated that the remaining 18 amino acids (CGRP 8 -37) are important for receptor recognition and docking. However, this sequence was found not to be involved in receptor activation. This finding led to the use of CGRPg-37 as a CGRP-R inhibitor.
  • the present invention provides compounds of the formula (I) represented by the fo wherein
  • A is a lipid anchor that promotes insertion of the compound into a plasma membrane;
  • L is a linker moiety of 1 nm to 50 nm in length; and
  • X is a modulator of the endosomal CGRP-R; wherein, in a preferred embodiment of the present invention, the lipid anchor partitions into lipid membranes that are insoluble in non-ionic detergent at 4°C; or a pharmaceutically acceptable salt thereof.
  • X is the CGRP-R antagonist olcegepant.
  • X is the CGRP-R antagonist CGRP 8 -37 and the compound of formula (Id) is represented by Compound 4:
  • the present invention provides a method for the treatment of a disease or disorder mediated by endosomal CGRP receptor signaling, comprising administering to a subject in need thereof an effective amount of a compound of formula (Id) as herein defined.
  • the present invention provides the use of a compound of formula (Id) as herein defined in the manufacture of a medicament for the treatment of a disease or disorder mediated by endosomal CGRP receptor signaling.
  • the present invention provides a compound of formula (Id) as herein defined for use in the treatment of a disease or disorder mediated by endosomal CGRP receptor signaling.
  • the disease or disorder mediated by endosomal CGRP receptor signaling is migraine and symptoms associated with migraine including pain, photophobia, phonophobia, nausea and vomiting, sensory disorders, pain including somatic pain and visceral pain, pain associated with cluster and chronic daily headache, respiratory disorders including COPD and asthma, gastrointestinal disorders including inflammatory bowel disease, irritable bowel syndrome, gastroparesis and functional dyspepsia, and chronic inflammatory disorders including osteoarthritis and rheumatoid arthritis.
  • the disease or disorder mediated by endosomal CGRP receptor signaling is migraine and its associated symptoms.
  • Lipid anchors of the formula (Ilia) having an amide, sulfonamide, urea or carbamate functional group at position 3 of the steroid structure can be prepared from 3- cholesteryla nine, for example, 3-cholesterylamine can be reacted with succinic anhydride in the presence of DMAP to afford the corresponding succinyl substituted compound.
  • the corresponding sulfonamide can be obtained by reaction of 3-cholesterylamine with chlorosulfonylacetic acid, which can be prepared as described in the literature (Hinman, R. L. and Locatell, L. J. Am. Chem. Soc. 1959, 81, 5655-5658).
  • the corresponding urea or carbamate can be prepared according to literature procedures via the corresponding isocyanate (Knolker, H.-J. T. et al, Angew. Chem. nt. Ed. 1995, 34, 2497; Knolker, H.-J. et al, Synlett 1996, 502;. Knolker, H.-J. and. Braxmeier, T. Tetrahedron Lett. 1996, 37, 5861).
  • Intermediates of compound (Ilia) having a phosphate or carboxymethylated phosphate at position 3 of the steroid structure can be prepared as described in the literature (Golebriewski, Keyes, Cushman, Bioorg. Med. Chem.
  • Lipid anchors of the formula (Ilia) having a thiol at position 3 of the steroid structure can be prepared as described in the literature (J. G. Parkes, J. G. et al., Biochim. Biophys. Acta 1982, 691 , 24-29), the corresponding carboxymethylated thiols are obtainable by simple alkylation as described for the corresponding amines and alcohols.
  • Lipid anchors of the formula (Ilia) having a difluoromethylenesulfone derivative at position 3 of the steroid structure can be prepared as described in the literature (Lapiene, J. et al., Bioorg. Med. Chem. Lett. 2004, 14, 151 - 155). Introduction of various side chains at position 17 of lipid anchors of the formula (Ilia) can be achieved by use of literature protocols starting from dehydroisoandrosterone or pregnenolone (Bergmann, E. D. et al., J. Am. Chem. Soc. 1959, 81 , 1239-1243 and references therein).
  • Lipid anchors of the formula (Ilia) which are derived from cholestane are obtainable from the corresponding precursors which are derived from cholesterol by reduction of the 5,6-double bond using literature protocols, e.g. hydrogenation in the presence of various transition metal catalysts.
  • Lipid anchors of the formula (Ila) having an oxygen derived substituent at position 3 are prepared in a similar manner as described for the lipid anchors of the formula (Ilia) starting from estrone.
  • Lipid anchors of the formula (Ila) having nitrogen derived substitution at position 3 can be prepared in a similar manner as described for lipid anchors of the formula (Ilia) starting from 3-amino estrone, which can be prepared as described in the literature (Zhang, X. and Sui, Z. Tetrahedron Lett. 2003, 44, 3071-3073; Woo, L. W. L. et al, Steroid Biochem. Molec. Biol. 1996, 57, 79-88).
  • Lipid anchors of the formula (Ila) having a sulfur derived substituent at position 3 can be prepared in a similar manner as described for lipid anchors of the formula (Ilia) starting from 3-thioestrone, which can be prepared as described in the literature (Woo, L. W. L. et al., J. Steroid Biochem. Molec. Biol. 1996, 57, 79-88). Introduction of various side chains at position 17 of the estrone structure can be achieved by a Wittig approach, followed by hydrogenation of the resulting double bond as described in the literature (Peters, R. H. et al., J. Org. Chem. 1966, 31 , 24-26). Further manipulations within the side chain (e.g.
  • Lipid anchors of the formula (Via) belonging to the class of ceramides, dehydroceramides and dihydroceramides with different hydrocarbon groups are obtainable as outlined in the literature (A. H. Merrill, Jr., Y. A. Hannun (Eds.), Methods in Enzymology, Vol. 31 1, Academic Press, 1999;. Koskinen, P. M and Koskinen, A. M. P. Synthesis 1998, 1075).
  • sphingosine base can be used as key intermediate for all lipid anchors of the formula (Via) having oxygen derived substitution at position 1 of the sphingosine backbone.
  • the corresponding amino derivatives are obtainable by substitution of the sulfonates, which can be prepared from the alcohols according to known protocols. Alkylation and acylation of 1 -amino or 1 -hydroxy derivatives can be achieved by reaction with bromo acetic acid and succinic anhydride, respectively.
  • the thioacetylated derivative can be prepared by substitution of a sulfonate with mercapto acetic acid. Phosphate and sulfate derivatives are obtainable as described in the literature (A. H.
  • Lipid anchors of the formula (Via) wherein R 5a is an amino or amino derived function can be prepared starting from sphingosine base, which is available as published by Koskinen (Koskinen, P. M. and Koskinen, A. M. P. Synthesis 1998, 1075), using standard protocols.
  • the corresponding 2- oxygen substituted sphingolipids can be obtained by a strategy published by Yamanoi (Yamanoi, T. et al., Chem. Lett. 1989, 335).
  • Lipid anchors of the formula (Via), wherein both R 8a represent a hydroxy group are obtainable by bishydroxylation of the corresponding alkene using known protocols.
  • the corresponding monohydroxy derivatives can be prepared as described in the literature (Howell, A. R. and Ndakala, A. J. Curr. Org. Chem. 2002, 6, 365-391). Modification of substituents R 6a and R 9a in lipid anchors of the formula (Via) can be achieved by protocols and strategies outlined in various review articles (Harwood, H. J. Chem. Rev. 1962, 62, 99-154; Gensler, W. J. Chem. Rev. 1957, 57, 191-280).
  • Lipid anchors of the formula (Vila) are obtainable by protocols described in the literature (Mrjller, S. et al, J. Prakt. Chem. 2000, 342, 779) and by combinations thereof with protocols described for the preparation of lipid anchors of the formula (Vila).
  • Lipid anchors of the formula (Villa), wherein R a and R a are oxygen derived substituents, can be prepared starting from commercially available (R)-(-)-2,2-dimethyl-l ,3-dioxolane- 4-methanol as outlined by Fraser-Reid (Schlueter, U. Lu, J. and Fraser-Reid, B. Org. Lett. 2003, 5, 255-257).
  • Lipid anchors of the formula (Villa), wherein R 4a and R 5a are nitrogen derived substituents are obtainable either starting from the corresponding oxygen substituted systems by nucleophilic replacement of the corresponding sulfonates and further modifications as outlined above, or starting from 1 ,2,3-triaminopropane which is obtainable as described in the literature (Henrick, K. et al., J Chem. Soc. Dalton Trans. 1982, 225-227).
  • Lipid anchors of the formula (IXa) are obtainable in a similar fashion as lipid anchors of the formula (Vila) or alternatively by ring closing metathesis of co-ethenylated intermediates of lipid anchors of the formula (Villa).
  • Lipid anchors of the formulae (Xa) and (XIa) are obtainable by synthetic strategies described in the literature (Xue, J. and Guo, Z. Bioorg. Med. Chem. Lett. 2002, 12, 2015- 2018; Xue, J. and Guo, Z. J. Am. Chem. Soc. 2003, 16334-16339; Xue, J. et al, J. Org. Chem. 2003, 68, 4020-4029; Shao, N., Xue, J. and Guo, Z. Angew. Chem. Int. Ed. 2004, 43, 1569-1573) and by combinations thereof with methods described above for the preparation of lipid anchors of the formulae (Via) and (Villa).
  • Lipid anchors of the formulae (Xlla), (XHIa) and (XIV a) are obtainable by total synthesis following synthetic strategies described in the literature (Knolker, H.-J. Chem. Soc. Rev. 1999, 28, 151 -157; Knolker, H.-J. and Reddy, K. R. Chem. Rev. 2002, 102, 4303-4427; Knolker, H.-J. and Knoll, J. Chem. Commun. 2003, 1170-1171 ; Knolker, H.-J. Curr. Org. Synthesis 2004, 1).
  • Lipid anchors of the formula (XV a) can be prepared by Nenitzescu-type indole synthesis starting from 4-methoxy-3-methylbenzaldehyde to afford 6-methoxy-5-methylindole.
  • Ether cleavage, triflate formation and Sonogashira coupling leads to the corresponding 6- alkynyl substituted 5-methylindole.
  • Nilsmeier formylation and subsequent nitromethane addition yields the 3-nitro vinyl substituted indole derivative which is subjected to a global hydrogenation resulting in the formation of the 6-alkyl substituted 5-methyltryptamine.
  • Acylation of the amino group using succinyl anhydride completes the preparation.
  • Known solid or solution phase techniques may be used in the synthesis of the peptides of the present invention, such as coupling of the N- or C-terminus to a solid support (typically a resin) followed by step-wise synthesis of the linear peptide.
  • a solid support typically a resin
  • Protecting group chemistries for the protection of amino acid residues, including side chains are well known in the art and may be found, for example, in: Theodora W. Greene and Peter G. M. Wuts, Protecting Groups in Organic Synthesis (Third Edition, John Wiley & Sons, Inc, 1999), the entire contents of which is incorporated herein by reference.
  • Methods for the preparation of compounds as described herein will be apparent to those skilled in the art and will comprise the steps of a) defining the distance between (a) phosphoryl head group(s) or an equivalent head group of the lipid anchor and a binding and/or interaction site of the modulator of an endosomal GPCR; b) selecting a linker which is capable of spanning the distance as defined in (a); and c) bonding the lipid anchor and the modulator of an endosomal GPCR by the linker as selected in (b).
  • yeast two or three hybrid systems peptide spotting, overlay assays, phage display, bacterial displays, ribosome displays), atomic force microscopy as well as spectroscopic methods and X-ray crystallography.
  • methods such as site-directed mutagenesis may be employed to verify deduced interaction sites of a given modulator of an endosomal GPCR or of a candidate modulator of an endosomal GPCR and its corresponding target.
  • the modulator of an endosomal GPCR is a molecule which is involved in biological processes which take place in endosomes and is mediated by a G protein- coupled receptor.
  • selection of a linker comprises the selection of linkers known in the art as well as the generation and use of novel linkers, for example, by molecular modelling and corresponding synthesis or further methods known in the art.
  • the term "spanning" as used herein with reference to step b) refers to the length of the linker selected to place the modulator of an endosomal GPCR at the correct locus on the a receptor when the lipid anchor forms part of the lipid layer of the endosome.
  • the linker moiety is to connect the lipid anchor to the modulator of an endosomal GPCR in order to allow the modulator of an endosomal GPCR to interact with the receptor when the lipid anchor is anchored in the endosome membrane.
  • the lipid anchor and the linker will contain functional groups allowing for the two to be covalently bonded.
  • the nature of the functional group of the lipid anchor is in no way limited and may include, for example, an amine group that forms an amide bond with the linker, or a hydroxyl or carboxylic acid group that forms and ether or ester bond with the linker.
  • the skilled addressee will understand that selection of the functional group at the end of the linker that connects with the endosomal GPCR will be dictated primarily by any available functional groups on the modulator for an endosomal GPCR of choice.
  • the functional group of the linker will comprise a complementary carboxylic acid or amine to form an amide bond.
  • the compounds of the present invention may exist in one or more stereoisomeric forms (e.g. diastereomers).
  • the present invention includes within its scope all of these stereoisomeric forms either isolated (in, for example, enantiomeric isolation), or in combination (including racemic mixtures and diastereomic mixtures).
  • the present invention contemplates the use of amino acids in both L and D forms, including the use of amino acids independently selected from L and D forms, for example, where the peptide comprises two alanine residues, each alanine residue may have the same, or opposite, absolute stereochemistry. Unless stated otherwise, the amino acid is taken to be in the L-configuration.
  • the invention thus also relates to compounds in substantially pure stereoisomeric form with respect to the asymmetric centres of the amino acid residues, e.g., greater than about 90% de, such as about 95% to 97% de, or greater than 99% de, as well as mixtures, including racemic mixtures, thereof.
  • diastereomers may be prepared by asymmetric synthesis, for example, using chiral intermediates, or mixtures may be resolved by conventional methods, e.g., chromatography, or use of a resolving agent.
  • chromatographic techniques such as high-performance liquid chromatography (HPLC) and reverse-phase HPLC may be used.
  • HPLC high-performance liquid chromatography
  • the compounds may be characterised by mass spectrometry and/or other appropriate methods.
  • the compound comprises one or more functional groups that may be protonated or deprotonated (for example at physiological pH) the compound may be prepared and/or isolated as a pharmaceutically acceptable salt. It will be appreciated that the compound may be zwitterionic at a given pH.
  • pharmaceutically acceptable salt refers to the salt of a given compound, wherein the salt is suitable for administration as a pharmaceutical. Such salts may be formed, for example, by the reaction of an acid or a base with an amine or a carboxylic acid group respectively.
  • Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids.
  • inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like.
  • organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • Pharmaceutically acceptable base addition salts may be prepared from inorganic and organic bases.
  • Corresponding counter ions derived from inorganic bases include the sodium, potassium, lithium, ammonium, calcium and magnesium salts.
  • Organic bases include primary, secondary and tertiary amines, substituted amines including naturally- occurring substituted amines, and cyclic amines, including isopropylamine, trimethyl amine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2- dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, and N-ethylpiperidine.
  • Acid/base addition salts tend to be more soluble in aqueous solvents than the corresponding free acid/base forms.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a tripartite compound as hereinbefore defined, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.
  • composition is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.
  • the compounds as hereinbefore described, or pharmaceutically acceptable salts thereof may be the sole active ingredient administered to the subject, the administration of other active ingredient(s) with the compound is within the scope of the invention.
  • a combination of two or more of the compounds of the invention will be administered to the subject.
  • the compound(s) could also be administered with one or more additional therapeutic agents in combination.
  • the combination may allow for separate, sequential or simultaneous administration of the compound(s) as hereinbefore described with the other active ingredient(s).
  • the combination may be provided in the form of a pharmaceutical composition.
  • combination refers to a composition or kit of parts where the combination partners as defined above can be dosed dependently or independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e., simultaneously or at different time points.
  • the combination partners can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
  • the ratio of the total amounts of the combination partners to be administered in the combination can be varied, e.g. in order to cope with the needs of a patient sub-population to be treated or the needs of the single patient which different needs can be due to age, sex, body weight, etc. of the patients.
  • the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the active compound care should be taken to ensure that the activity of the compound is not destroyed in the process and that the compound is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the compound by means known in the art, such as, for example, micro encapsulation. Similarly the route of administration chosen should be such that the compound reaches its site of action.
  • Those skilled in the art may readily determine appropriate formulations for the compounds of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or sulphite, and metal chelators such as EDTA.
  • phenolic compounds such as BHT or vitamin E
  • reducing agents such as methionine or sulphite
  • metal chelators such as EDTA.
  • the compounds as hereinbefore described, or pharmaceutically acceptable salts thereof, may be prepared in parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery.
  • the pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage and may be preserved against reduction or oxidation and the contaminating action of microorganisms such as bacteria or fungi.
  • the solvent or dispersion medium for the injectable solution or dispersion may contain any of the conventional solvent or carrier systems for the active compound, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • agents to adjust osmolality for example, sugars or sodium chloride.
  • the formulation for injection will be isotonic with blood. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.
  • Sterile injectable solutions are prepared by incorporating the compounds of the invention in the required amount in the appropriate solvent with various of the other ingredients such as those enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • preferred methods of preparation are vacuum drying or freeze-drying of a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.
  • compositions include oral and enteral formulations of the present invention, in which the active compound may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, com starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the compounds of the invention may be incorporated into sustained-release preparations and formulations, including those that allow specific delivery of the active peptide to specific regions of the gut.
  • Liquid formulations may also be administered enterally via a stomach or oesophageal tube.
  • Enteral formulations may be prepared in the form of suppositories by mixing with appropriate bases, such as emulsifying bases or water-soluble bases. It is also possible, but not necessary, for the compounds of the present invention to be administered topically, intranasally, intravaginally, intraocularly and the like.
  • Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active ingredient may be compounded for convenient and effective administration in therapeutically effective amounts with a suitable pharmaceutically acceptable vehicle in dosage unit form.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.25 ⁇ g to about 2000 mg. Expressed in proportions, the active compound may be present in from about 0.25 ⁇ g to about 2000 mg/mL of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the term "effective amount" refers to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur once, or at intervals of minutes or hours, or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. A typical dosage is in the range of 1 ⁇ g to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage.
  • the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.
  • treatment and “treating” as used herein cover any treatment of a condition or disease in an animal, optionally a mammal, like a human, and includes treating any disease or disorder that is mediated by endosomal GPCR signaling.
  • prevention and “preventing” as used herein cover the prevention or prophylaxis of a condition or disease in an animal, optionally a mammal, like a human and includes prevention of a disease or disorder that is mediated by endosomal GPCR signaling.
  • Example 1 Preparation procedures of the tripartite compounds.
  • tripartite compounds according to the present invention have been prepared by the following experimental procedures:
  • Fmoc deprotection was achieved using 20% piperidine in N,N-dimethylformamide (DMF).
  • Fmoc-PEG12-OH (2 eq) was coupled to resin-bound NH 2 -Asp(OChol) with PyBOP (2 eq) and DIPEA (3 eq) in DCM.
  • Fmoc deprotection was achieved using 20% piperidine in N,N-dimethylformamide (DMF).
  • the NK1R antagonists were coupled to the spacer-lipid conjugate on resin.
  • the construct was then cleaved from resin using 95% trifluoroacetic acid and purified by silica gel chromatography (DCM:MeOH, 98:2 to 80:20), providing the tripartite probe Compound B as a viscous oil (34.8 mg).
  • the de-Boc material (150 mg, 0.303 mmol) was coupled to N-Boc isonipecotic acid (76 mg, 0.334 mmol) with l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5- )]- pyridinium 3-oxide hexafluorophosphate (HATU) H(127 mg, 0.334 mmol), in DMF (3 mL) with activation in situ using DIPEA (158 ⁇ , 0.909 mmol) for 3h. The mixture was diluted with EtOAc and washed with water the saturated bicarbonate and finally brine.
  • HATU 3-oxide hexafluorophosphate
  • the construct was then cleaved from resin using 95% trifluoroacetic acid and purified by reverse-phase high-performance liquid chromatography (HPLC) (Phenomenex Luna C8 column, Lane Cove, Australia) with 0.1% TFA/H 2 0 and 0.1% TFA/ACN as solvents, providing the tripartite probe Compound A as a viscous oil (30.2 mg).
  • HPLC reverse-phase high-performance liquid chromatography
  • the following compounds can be made using the same chemistry and starting from the indicated NK1 antagonist.
  • the acid D-3 (119 mg) was coupled to resin-bound NH 2 -PEG12-Asp(OChol)-resin (300 mg) with PyBOP (2 eq) and DIPEA (6 eq) in DCM:DMF (1 : 1) for 2h.
  • the construct was then cleaved from resin using 95:5 TFA:TIPS, concentrated to a esidue that was taken up in DCM and washed with bicarbonate, dried (MgSC ⁇ ), filtered, and concentrated to a residue (118 mg) and purified by silica gel chromatography (MeOH:EtOAc:Et 2 NH 10:90: 1) providing Compound D as a clear colourless viscous oil (68 mg, 69%).
  • Endosomal NK R signaling generates subcellular signals that underlie neuronal activation and hyperalgesia. Painful and pro-inflammatory stimuli, including the transient receptor potential vanilloid 1 agonist capsaicin, stimulate substance P (SP) release from primary sensory nociceptors in laminae I, II of the dorsal horn, where SP stimulates NKiR endocytosis in spinal neurons.
  • SP substance P
  • a tripartite probe based on the present invention can be synthesised comprising the lipid cholestanol or ethyl ester (negative control, no membrane anchoring), a polyethylene glycol linker and the fluorescent dye Cyanine 5 (Cy5), in addition to the tripartite compounds of the present invention.
  • Cy5-cholestanol After incubation with HEK293 cells, Cy5-cholestanol will insert into the plasma membrane by 5 min and can be detected in endosomes by 30-60 min by which time >50% of probe will be internalized, whereas the Cy5-ethyl ester will remain in the extracellular fluid. After 4 h, Cy5-cholestanol will redistribute from the plasma membrane to Rab5a- positive early endosomes containing NKiR-GFP.
  • a FRET assay can be used to quantitatively assess the capacity of a tripartite probe to target the NKiR in endosomes as follows:
  • HEK293 cells are transfected with 50 ng/well of NKiR with extracellular N-terminal Snap- Tag®. After 48 h, the cell surface NKiR is labeled with SNAP-SurfaceTM 549 photostable fluorescent substrate (New England Biolabs) (1 ⁇ , 30 min, 37°C in DMEM, 0.5% BSA). Cells were washed, recovered in DMEM for 30 min, and stimulated with substance P (SP) (10 nM, 30 min, 37°C) to induce NKiR endocytosis. Cells are incubated with Cy5-cholestanol (200 nM, 37°C).
  • SP substance P
  • SNAP-549/Cy5 sensitized emission FRET is analysed by confocal microscopy using sequential excitation with Argon (514 nm)/HeNe (633 nm) lasers and emission at 570-620 nm (SNAP-549 donor) and 670-750 nm (Cy5 FRET and Cy5 acceptor) before and after addition Cy5-cholestanol.
  • Controls include non-transfected HEK293 cells (acceptor only) and HEK293 cells not treated with Cy5-cholestanol (donor only).
  • FRET is analysed as described and expressed as emission ratios relative to controls (F/F 0 ).
  • NKiR with extracellular N-terminal Snap-Tag can be expressed in HEK293 cells and cell- surface NKiR is labelled with non-membrane permeant SNAP-SurfaceTM 549 photostable fluorescent substrate. SP (10 nM, 30 min) evokes translocation of all detectable SNAP-549 to endosomes.
  • SNAP-549-NKiR and Cy5-cholestanol FRET can be detected after adding Cy5- cholestanol, and increased for >75 min. Analysis of the subcellular origin of FRET can then confirm that most of the signal will be intracellular, proving probe association with endosomal NKiR.
  • tripartite compound of the present invention When cells are preincubated with a tripartite compound of the present invention, e.g. for 30 min, and then immediately challenged with SP, suitable antagonists will be shown to block activation of nuclear and cytosolic ERK indicating effective antagonism of cell-surface NKiR. In contrast, when cells are pulse incubated with antagonists for 30 min, washed and then stimulated with SP 4 h later, the tripartite compound alone will inhibit nuclear ERK and none of the antagonists inhibit cytosolic ERK.
  • the tripartite compound of the present invention does not affect isoprenaline-induced activation of nuclear ERK, which is mediated by the endogenous 2-adrenergic receptor.
  • conjugation to cholestanol delivers probes to NKiR-positive early endosomes, and after pulse incubation the tripartite compounds of the present invention are capable of causing sustained and selective antagonism of the endosomal not cell-surface NKiR.
  • slices of rat spinal cord can be incubated with the tripartite compound of the present invention to assess whether antagonism of the endosomal NKiR blocks sustained SP-induced excitation of spinal neurons.
  • an endosomally-targeted and membrane-anchored KjR antagonist effectively blocks persistent SP-induced activation of spinal neurons and causes long-lasting analgesia.
  • intrathecal injections of Cy5-cholestanol, spantide or the tripartite compound of the present invention can be made to mice. After 3 h, capsaicin is injected into the paw. The tripartide compound of the present invention, but not spantide or Cy5-cholestanol will markedly inhibit capsaicin-evoked hyperalgesia. When administered 30 min after capsaicin, intrathecal spantide will have a small and transient analgesic effect, whereas the tripartite compound will cause a delayed, persistent and substantial inhibition analgesia.
  • Cy5-cho3estanol will be detected in laminae I-III neurons after 6 h, suggesting effective probe delivery.
  • the tripartite compound When injected intrathecally 3 h before intraplantar formalin, the tripartite compound will markedly inhibit both phases of nocifensive behavior more effectively than spantide.
  • the tripartite compound When injected intrathecally 36 h after intraplantar CFA, the tripartite compound is analgesic after 4 h, and analgesia is sustained for >6 h, whereas the analgesic actions of spantide will be minor and transient.
  • the small molecule N 3 R antagonist L733-0660 can be also tested by conjugation to cholestanol via a polyethylene glycol linker. When injected intrathecally 3 h before intraplantar capsaicin, this conjugate will show sustained analgesia for > 4h, whereas the effect of L-733,060 is only small and transient.
  • an endosomally-targeted and membrane-anchored N iR antagonist effectively blocks persistent SP-induced activation of spinal neurons and causes long-lasting analgesia.
  • spantide and the tripartite compound are similarly stable in human CSF for > 4h, after intrathecal injection to mice spantide will be extensively metabolized whereas the tripartite compound is stable. Endosomal targeting and retention may contribute to the m vivo stability.
  • Parasagittal slices (340-400 ⁇ ) are prepared using a vibratome from the lumbar region of the rat spinal cord in ice-cold sucrose-based artificial CSF (sACSF) (mM: 100 sucrose, 63 NaCl, 2.5 KC1, 1.2 NaH 2 P0 4 , 1.2 MgCl 2 , 25 glucose, 25 NaHC0 3 ; 95% 0 2 /5% C0 2 ).
  • sACSF sucrose-based artificial CSF
  • NMDG N-Methyl-D-Glucamine
  • rACSF recovery ACSF
  • Slices are transferred to normal ACSF (mM: 125 NaCl, 2.5 KC1, 1.25 NaH 2 P0 4 , 1.2 MgCl 2 , 2.5 CaCl 2 , 25 glucose, 11 NaHC0 3 ; 95% 0 2 /5% C0 2 ) containing 10 ⁇ MK- 801 (45 min, 34°C), then maintained at RT.
  • Slices of rat spinal cord are transferred to a recording chamber and superfused with normal ACSF (2 ml.min "1 , 36°C).
  • Dodt-contrast optics are used to identify large (capacitance > 20 pF), putative NKiR-positive neurons in lamina I based on their position, size and fusiform shape with dendrites that are restricted to lamina I.
  • Slices are preincubated with Dy4 or Dy4 inact (both 30 ⁇ , 0.03% DMSO) for 10 min before recording, or are preincubated with the tripartide compound of the present invention and spantide, (both 1 ⁇ in 0.01% DMSO) for 60 min, washed and incubated in antagonist-free ACSF for a further 60 min before recording.
  • PS2 will not be soluble in the low concentrations of DMSO required for electrophysiology.
  • NKiR-positive, presumed nociceptive lamina I neurons are visually identified as described (Imlach, W. L., et al., Molecular Pharmacology, 2015, 88, 460-428).
  • Spontaneous currents are recorded in cell-attached configuration (Multiclamp 700B, Molecular Devices, Sunnyvale, CA), sampled at 10 kHz, high pass filtered at 1 Hz, and capacitatively coupled action potential events are analyzed using Axograph X, V 1.4.4 (Axograph).
  • Patch electrodes should contain KMES-based internal solution (mM: 105 KMES, 20 NaCl, 5 HEPES, 10 BAPTA, 1.5 MgCl 2 , 4 MgATP, 0.4 NaGTP, 0.1% biocytin; 285-295 mosmol.l "1 ) to facilitate subsequent recordings of action potential properties in whole-cell configuration.
  • KMES-based internal solution mM: 105 KMES, 20 NaCl, 5 HEPES, 10 BAPTA, 1.5 MgCl 2 , 4 MgATP, 0.4 NaGTP, 0.1% biocytin; 285-295 mosmol.l "1 ) to facilitate subsequent recordings of action potential properties in whole-cell configuration.
  • CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) (10 ⁇ ; AMPA/kainate receptor antagonist), picrotoxin (100 ⁇ ; GABAA receptor antagonist), strychnine (0.5 ⁇ ; glycine and acetylcholine receptor antagonist), and AP5 ((2i?)-amino-5- phosphonovaleric acid; (2i?)-amino-5-phosphonopentanoate) (100 ⁇ ; NMDA receptor antagonist) to minimize presynaptic influences on action potential properties.
  • Slices are then challenged by superfusion with SP (1 ⁇ ) for 2 min.
  • NKiR endocytosis spinal cord slices (400 ⁇ ) are incubated with SP (1 ⁇ , 5 min), fixed in PFA (4h, RT), cryoprotected, and are processed to localize the NKiR.
  • mice are acclimatized to the experimental apparatus and environment for 1-2 h on 2 successive days before experiments. Mechanical hyperalgesia is assessed by paw withdrawal to stimulation of the plantar surface of the hind-paw with graded von Frey filaments. On the day before the study, von Frey scores are measured in triplicate to establish a baseline for each animal. To assess edema of the paw, hindpaw thickness is measured using digital calipers before and after treatments. For intraplantar injections, mice are sedated (5% isoflurane).
  • Capsaicin (5 ⁇ g), Complete Freund's Adjuvant (CFA, 2 mg/rnl), or vehicle (capsaicin, 20% ethanol, 10% Tween 80, 70% saline; CFA, saline) is injected subcutaneously into the plantar surface of the left hindpaw (10 ⁇ ).
  • CFA Complete Freund's Adjuvant
  • CFA saline
  • mice are placed in a Perspex container and nocifensive behavior (flinching, licking, biting of the injected paw) is recorded for 60 min. The total number of nocifensive events is subdivided into acute (I, 0-10 min) and tonic (II, 10-60 min) phases. At the end of experiments, mice are transcardially perfused with PBS and PFA, and the spinal cord is removed and processed to localize the NKiR by immunofluorescence, as described for rats. Investigators are unaware of test agents.
  • Intrathecal injections (5 ⁇ , L3/L4) are made into conscious mice.
  • Dy4 inact, PS2, PS2 inact (all 50 ⁇ ), SR-140333 (15 ⁇ ), SM-19712 (8 mM), U0126 (100 ⁇ ), or vehicle (1% DMSO/saline) is injected intrathecally 30 min before intra-plantar injection of capsaicin or formalin, or 36 h after CFA.
  • Fmoc-PAL-PEG-PS resin for SPPS was obtained from Applied Biosciences.
  • PEG amino acids Fmoc-PEG3-OH and Fmoc-PEG4-OH
  • Fmoc-Asp(OChol)-OH and Fmoc-Asp(OEt)-OH were synthesized from commercially-available Fmoc-Asp-0/-Bu using standard chemistry (side-chain esterification, followed by acid-deprotection of the C-terminus). All other Fmoc amino acids (with suitable protecting groups) were obtained from Chem-Impex or Auspep.
  • Coupling agents HBTU, HCTU
  • solvents were used as received.
  • LC-MS Liquid chromatography-mass spectrometry
  • Preparative high-pressure liquid chromatography was carried out using a Waters Prep LC system consisting of a solvent pump, system controller and UV absorbance detector, using a Phenomenex Luna C8 (2) 10 ⁇ column (10 A, 50 ⁇ 21.2 mm) for larger-scale preparative purifications and a Phenomenex Luna C8 (2) 5 ⁇ column (100 A, 250 ⁇ 10 mm) for smaller-scale semi-preparative purifications. All separations used buffers A and B consisting of 0.1% TFA/H 2 0 and 0.1% TFA/ACN, respectively as solvent.
  • the protected linear peptide D Arg-Pro-Lys-Pro-Gln-Gln- D Trp-Phe- D Trp-Leu-Leu-NH 2 (Spantide I) was synthesised by automated SPPS as described above starting with N- deprotection of Fmoc-PAL-PEG-PS resin (278 mg, 0.18 mmol/g, 50.0 ⁇ ). After the final N-deprotection step, a portion of the material was cleaved from resin to give a crude residue that was suspended in a 1 : 1 mixture of ACN/H 2 0 containing 0.1% TFA to ensure complete side-chain deprotection.
  • N-deprotected resin bound compound (375 mg, 50.0 ⁇ ) was transferred a 5 mL filtration column and subjected to manual SPPS with Fmoc-PEG4-OH, Fmoc-PEG3-OH, Fmoc-PEG4-OH and Fmoc-Asp(OChol)-OH, respectively.
  • the N-terminus of the peptide was capped by exposure of the resin to a solution of DIPEA (100 ⁇ ) and acetic anhydride (1.5 mL) in DMF (1.5 mL) with continuous agitation for two hours. The resin was then washed in the usual way and the peptide cleaved from resin to give the crude product. Purification by preparative HPLC (50-100% Buffer B over 35 minutes) gave Compound 1 (5.5 mg).
  • Synthesis was carried out by manual SPPS as described above, starting with Fmoc-PAL- PEG-PS resin (loading 0.17 mmol/g from Life Technologies) on a 0.025 mmol scale (147 mg of resin). The coupling/deprotection cycle was repeated a total of 4 times, using Fmoc- Asp(OChol)-OH, Fmoc-PEG4-OH, Fmoc-PEG3-OH and Fmoc-PEG4-OH, respectively.
  • the bromo-terminated resin bound compound was reacted with the NKiR antagonist L-733,060 HC1 (0.8 mol eq.) in a solution of tetra-n- butylammonium iodide (TBAI, 0.5 mol eq.) and DIPEA (5 mol eq.) of over the course of 3 days.
  • TBAI tetra-n- butylammonium iodide
  • DIPEA 5 mol eq.
  • the product was cleaved from the resin using 2 mL of a mixture of TFA/TIPS/H 2 0 (95:2.5:2.5) for 1.5 h, after which time the resin was filtered, washed with 2 mL of TFA, and the solvent removed under a stream of nitrogen. The crude residue was then taken up in a mixture of milli-Q water (1 mL) and acetonitrile (0.5 mL) and subjected to RP-HPLC purification. Compound 2 was confirmed as having the correct molecular weight by ESI-MS analysis: m/z calculated: 864.53 for C 9 oHi 44 F 6 N 6 Oi 9 [M + 2H] 2+ ; observed: m/z 864.70.
  • NKiR agonist GR73632 [Ava-Phe-Phe-Pro-N-MeLeu-Met-NH 2 ] was prepared by automated SPPS starting with N-deprotection of Fmoc-PAL-PEG-PS resin as described above. After final N-deprotection, a portion of the material was cleaved from the resin using TFA solution (95:2.5:2.5 TFA:TIPS:H20) to give the desired agonist peptide. The product was confirmed by mass spectrometry: m/z 766.4, calcd for C40H59N7O6S [M + H ] m/z 766.4.
  • Compound 3 was derived from a portion of resin-bound GR73632.
  • the N-deprotected GR73632 resin was subjected to manual SPPS using Fmoc-PEG4-OH, Fmoc-PEG3-OH, Fmoc-PEG4-OH and Fmoc-Asp(OChol)-OH as the amino acids.
  • the N-terminus of the peptide was capped by exposure to DIPEA and AC 2 O in DMF for 2 h.
  • the construct was cleaved from the resin as above and purified by repeated HPLC.
  • Compound 3 m/z 996.6, calcd for C104H173N11O24S [M + 2H + ] m/z 996.1.
  • Synthesis of the protected linear peptide VTHRLAGLLSRSGGVVKDNFVPTNVGSEAF- NH 2 (CGRP8-37) was carried out via manual SPPS as explained above. Synthesis started with NovaSyn®TG R R resin (loading 0.18 mmol/g) on a 0.18 mmol scale (1 g of resin). A small portion of the completed CGRPg-37 peptide on resin (100 mg) was cleaved, filtered and washed with 2 mL of the cleavage solution, and solvent was removed under a stream of nitrogen. The crude peptide was subjected to RP-HPLC purification providing CGRPg. 37 as a white amorphous solid in a yield of 7 mg.
  • Bioluminescence Resonance Energy Transfer was employed to quantitatively assess the proximity of the NK R to key regulators of GPCR trafficking (Parrs), and resident proteins of plasma (KRas) and early endosomal (Rab5a) membranes in HEK293 cells (Fig. 1A).
  • Substance P SP, 1, 10 nM
  • SP stimulated BRET between NKiR-RLuc8 and arrl-YFP and Parr2-YFP
  • SP decreased NKiR-RLuc8/KRas-Venus BRET and concomitantly increased NKiR-RLuc8/Rab5a-Venus BRET (Fig. ID), consistent with NKiR endocytosis.
  • NKiR was localised by studying trafficking of Alexa568-SP in HEK-NKiR cells.
  • Alexa568-SP bound to the plasma membrane receptor at 4°C, and completely redistributed to endosomes after 30 min at 37°C.
  • Dy4 and PS2 but not inactive controls, caused retention of Alexa568-SP in vesicles close to the plasma membrane (data not shown).
  • SP promotes NK R association with arrs, and induces clathrin- and dynamin-dependent endocytosis of the NKiR.
  • FRET Resonance Energy Transfer
  • SP did not affect plasma membrane PKC activity (not shown) but caused a rapid and sustained increase in cytosolic PKC activity (Fig. 2D).
  • SP transiently increased plasma membrane cAMP (not shown) and caused a sustained increase in cytosolic cAMP (Fig. 2F).
  • PS2 and Dy4 but not inactive controls, abolished SP stimulation of nuclear ERK (Fig. 2B), cytosolic PKC (Fig. 2D) and cytosolic cAMP (Fig. 2F).
  • PS2 and Dy4 did not suppress SP activation of cytosolic ERK or plasma membrane cAMP, but caused both responses to become sustained.
  • PS2 and Dy4 amplified plasma membrane PKC activity.
  • Dynamin WT magnified SP activation of nuclear ERK, whereas dynamin K44E abolished activation of nuclear ERK (Fig. 2G).
  • Dynamin K44E did not block activation of cytosolic ERK but caused the response to become sustained.
  • Dynamin and clathrin siRNA inhibited expression and prevented SP activation of nuclear ERK (Fig. 2H).
  • clathrin- and dynamin-dependent NKiR endocytosis is required for activation of nuclear ERK, which also depends on arrs and for activation of cytosolic PKC and cAMP.
  • the G q inhibitor UBO-QIC prevented SP activation of nuclear ERK (Fig. 3E), which also depends on arrs and PKC and is independent of epidermal growth factor receptor transactivation.
  • UBO-QIC, the PLC inhibitor U73122 and the Ca 2+ chelator EGTA prevented activation of cytosolic PKC (Fig. 3F), consistent with activation of the G q , PLC and Ca 2+ -dependent PKC pathway.
  • UBO-QIC did not affect NKiR endocytosis. These results suggest that activated NKiR and G q translocate to endosomes, where Ga q generates nuclear ERK and cytosolic PKC and
  • Painful and proinflammatory stimuli including capsaicin (transient receptor potential vanilloid 1 agonist), trigger SP release from primary sensory nociceptors in laminae I, II of the dorsal horn, where SP stimulates NKiR endocytosis in spinal neurons 10 ' 11 . SP causes persistent NKiR-dependent excitation of spinal neurons 20 .
  • cell-attached patch clamp recordings were made from NKiR-positive neurons in lamina I of the dorsal horn in slices of rat spinal cord. SP (1 ⁇ , 5 min) stimulated NKiR endocytosis, that was prevented by Dy4 (Fig. 4A).
  • Dy4 did not affect glutaminergic primary afferent synaptic transmission or synaptic rundown in slices of rat spinal cord (not shown). PS2 and Dy4 did not affect basal or capsaicin-stimulated release of SP-IR and CGRP-IR from segments of mouse dorsal spinal cord.
  • Example 7 N3 ⁇ 4R Endocytosis and Pain Transmission.
  • NKiR-IR was mostly at the plasma membrane of the soma and neurites of lamina I neurons (80.7 ⁇ 1.6% of total NKiR-IR within 0.5 ⁇ of the soma plasma membrane).
  • NKiR endocytosis (42.1 ⁇ 5.6% NKiR-IR at plasma membrane, PO.001 to control; Fig. 4A).
  • Dy4 or PS2, but not Dy4 inact or PS2 inact inhibited capsaicin-stimulated NKiR endocytosis (% membrane: Dy4 59.6 ⁇ 0.2, Dy4 inact 49.9 ⁇ 0.8, O.001; PS2 69.0 ⁇ 1.1, PS2 inact 51.9 ⁇ 1.3, PO.05; Fig. 4A).
  • NKiR-expressing spinal neurons Painful peripheral stimuli activate ERK in NKiR-expressing spinal neurons, which coincides with receptor endocytosis and contributes to hyperalgesia.
  • Intraplantar capsaicin stimulated ERK phosphorylation in lamina I dorsal horn neurons (Fig. 5B).
  • Dy4 or PS2 but not the inactive analogues, prevented capsaicin-evoked activation of spinal ERK.
  • capsaicin stimulates clathrin- and dynamin-dependent NKiR endocytosis and ERK activation in spinal neurons in vivo.
  • mice C57BL/6, male, 6-10 weeks were injected intrathecally (L3/L4) with vehicle, NKiR antagonist SR140,333, Dy4, Dy4 inact, PS2 or PS2 inact. After 30 min, intraplantar injections of vehicle or capsaicin were made into the left hindpaw. Mechanical hyperalgesia was measured by stimulation of the plantar surface of the left and right paws with von Frey filaments, and inflammatory edema was assessed by measuring left paw thickness with calipers. In vehicle (intrathecal)-treated mice, capsaicin caused mechanical hyperalgesia and edema of the left paw.
  • Intrathecal arr 1+2 siRNA reduced spinal levels of arrl and 2 and also inhibited capsaicin-evoked hyperalgesia at 36 h (Fig. 5G-H). siRNAs did not affect paw withdrawal responses of the uninjected right hind paw (not shown).
  • Intrathecal MEK inhibitor U0126 inhibited capsaicin-evoked hyperalgesia (Fig. 61).
  • Intrathecal Dy4 and PS2 inhibited both phases of nocifensive behavior in mice evoked by intraplantar injection of formalin (Fig. 6J, K).
  • Intraplantar injection of complete Freund's adjuvant (CFA) caused sustained mechanical hyperalgesia in mice, that was partially reversed by intrathecal Dy4 and PS2 within 1-5 h (Fig. 6L).
  • Intraplantar capsaicin, formalin and CFA all evoked NKiR endocytosis in neurons of mouse dorsal horn, that was inhibited by intrathecal Dy4 (data not shown).
  • a tripartite probe was synthesised comprising the lipid cholestanol or ethyl ester (control, no membrane anchoring), a polyethylene glycol linker and the fluorescent dye Cyanine 5 (Cy5), in addition to the compounds of the invention.
  • Cy5-cholestanol After incubation with HEK293 cells, Cy5-cholestanol inserted into the plasma membrane by 5 min and was detected in endosomes by 30-60 min by which time >50% of probe was internalized, whereas Cy5-ethyl ester remained in the extracellular fluid (Fig. 7B). After 4 h, Cy5- cholestanol had redistributed from the plasma membrane to Rab5a-positive early endosomes containing NKiR-GFP.
  • NKiR with extracellular N-terminal Snap-Tag was expressed in HEK293 cells and cell-surface NKiR was labelled with non-membrane permeant SNAP-SurfaceTM 549 photostable fluorescent substrate. SP (10 nM, 30 min) evoked translocation of all detectable SNAP-549 to endosomes.
  • SNAP-549-NKiR and Cy5-cholestanol FRET was detected within 5 min of adding Cy5-cholestanol, and increased for >75 min (data not shown). Analysis of the subcellular origin of FRET confirmed that >75% of the signal was intracellular, consistent with probe association with endosomal NKiR
  • Compound 1 antagonized SP (3 nM, EC 8 o)-stimulated Ca 2+ signaling in HEK-NKiR cells
  • the small molecule N 3 R antagonist L733-0660 was conjugated to cholestanol via a polyethylene glycol linker (Compound 2), When injected intrathecally 3 h before intraplantar capsaicin, Compound 2 showed sustained analgesia for > 4h, whereas the effect of L-733,060 was small and transient (Fig. 8J).
  • an endosomally-targeted and membrane-anchored N i antagonist effectively blocks persistent SP-induced activation of spinal neurons and causes long-lasting analgesia.
  • spantide and Compound 1 were similarly stable in human CSF for > 4h, after intrathecal injection to mice spantide was extensively metabolized whereas Compound 1 was stable (not shown). Endosomal targeting and retention may contribute to the in vivo stability of spantide-cholestanol.
  • CGRP and SP can be co-released from primary sensory neurons to evoke pain and neurogenic inflammation.
  • CGRP J nM
  • CGRP J nM
  • CLR-RLuc8/KRas- Venus BRET CLR-RLuc8/Rab5a-Venus BRET
  • PS2 and dynaminK44E inhibited these effects.
  • CGRP stimulated nuclear and cytosolic ERK.
  • Dy4 and dynaminK44E prevented activation of nuclear but not cytosolic ERK.
  • Compound 4 comprising the CLR antagonist CGRP 8 -37 was found to effectively antagonise CGRP-stimulated Ca 2+ signaling in HEK-CLR/RAMP 1 cells. After preincubation with HEK293 cells for 30 min, both CGRP 8"37 and Compound 4 inhibited CGRP-stimulated activation of nuclear and cytosolic ERK (data not shown). When cells were pulse incubated with antagonists for 30 min, washed and then stimulated with CGRP 4 h later, Compound 4 alone strongly inhibited nuclear ERK and neither antagonist strongly inhibited cytosolic ERK.
  • GR73632 [Ava-Phe-Phe-Pro-N-MeLeu-Met-Nty is a neurokinin 1 receptor agonist that is not susceptible to cleavage by the endothelin converting enzyme 1 (Hagan R.M. et al, Neuropeptides 1991 , 19, 127-35). GR73632 was conjugated to cholestanol (Compound 3) or ethyl ester (GR73632-ethyl ester) via a flexible PEG linker. The effect of these compounds in HEK293 cells expressing NK1R was then studied.
  • FRET Forster resonance energy transfer
  • EKAR extracellular- regulated kinase activity reporter
  • ERK extracellular signal related kinase activation
  • Preliminary data in FRET assays imaged over 20 minutes showed that the free GR73632 agonist stimulated sustained cytosolic and nuclear ERK phosphorylation whereas no ERK phosphorylation was observed in response to the GR73632-ethyl ester.
  • Compound 3 stimulates sustained nuclear ERK phosphorylation but no cytosolic ERK.
  • AlexaFluor568 NHS ester (Invitrogen, Carlsbad, CA; 6.3 ⁇ ) was incubated with SP (1.48 ⁇ ) and triethylamine (2.9 ⁇ ) in DMF (500 ⁇ ) (15 h, room temperature, RT). Alexa568-SP was purified by reverse-phase HPLC. The product was confirmed by mass spectrometry: m/z 1012.8, calcd for C 96 H127N1 9 O24S 3 [M + 2H + ] m/z 1012.9. cDNAs.
  • BRET probes NKiR-RLuc8, KRas-Venus, Rab5a-Venus, arrl-YFP, arr2-YFP, Ga q - RLuc8 and Gy2-Venus have been described (Kocan, M., et al, Frontiers in Endocrinology, 2010, 1, 12; Lan, T. H., et al, Traffic, 2012, 13, 1450-1456).
  • CytoEKAR and NucEKAR and CytoCKAR and pmCKAR were from Addgene (plasmids 18680, 18681, 14870, 14862, respectively).
  • CytoEpac2-camps was from M Lohse (University of Wurzburg) and pmEpac2-camps was from D Cooper (University of Cambridge).
  • GFP-dynamin and GFP- dynamin K44E have been described (Schmidlin, F. et al, J. Biol Chem, 2001, 276, 25427- 25437).
  • Human NK R with extracellular N-terminal Snap-Tag® was from Cisbio. Full length and truncated 5312 rat HA-NKiR have been described (Dery, O., Defea, K. A. & Bunnett, N. W., American Journal of Physiology, 2001, 280, CI 097-1106).
  • RLuc8 fusions of these constructs were generated by removal of the stop codon by PCR and subcloning into a pcDNA3.1-RLuc8 vector.
  • HA-CLR and Myc-RAMPl have been described (Cottrell, G. S. et al, J Biol. Chem., 2007, 282, 12260-12271).
  • CLR-RLuc was from M. Bouvier (Universite de Montreal).
  • HEK293 cells stably expressing rat NK R with N-terminal HA11 epitope have been described (Roosterman, D. et al. P.N.A.S., 2007, 104, 11838-11843) .
  • HEK293 cells were transiently transfected using polyethylenimine (Polysciences) or FuGene (Promega). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS (37°C, 5% C0 2 ).
  • DMEM Dulbecco's modified Eagle's medium
  • HEK293 cells were transfected with the following cDNAs: 1 ⁇ g NKiR-RLuc8 or 1 ⁇ g CLR-RLuc8 + 1 ⁇ g Myc-RAMPl + 4 ⁇ g KRas-Venus, 4 ⁇ g Rab5a-Venus, 4 ⁇ g arrl- YFP, or 4 ⁇ g arr2-YFP; or 1 ⁇ g NKiR + 0.5 ⁇ g Ga q -RLuc8 + 1 ⁇ g ⁇ + 4 ⁇ g Gy 2 - Venus; or 1 ⁇ g NKiR + 0.5 ⁇ g Go q -RLuc8 + 1 ⁇ g ⁇ + 1 ⁇ g Gy 2 + 4 ⁇ g Rab5a-Venus.
  • HBSS Hank's balanced salt solution
  • RLuc substrate coelenterazine h 5 ⁇ , 15 min.
  • BRET ratios were determined using a microplate reader LUMIstar Omega (BMG LabTech) before and after challenge with SP (0.1-10 nM) or vehicle (dH 2 0).
  • HEK293 cells were transfected with 55 ng/well rat NKiR with N-terminal HA.11 epitope tag (HA-NKiR) or CLR plus RAMP1 and 40 ng/well FRET biosensors. FRET was assessed 48 h after transfection, following serum restriction (0.5% FBS overnight). For experiments using clathrin or dynamin siRNA, cells were transfected with 55 ng/well rat HA-NKiR, 40 ng/well FRET biosensor and 25 nM/well scrambled, clathrin or dynamin ON-TARGETplus SMARTpool siRNA (GE Dharmacon). FRET was assessed 72 h after transfection, following serum restriction (0.5% FBS overnight).
  • Cells were equilibrated in HBSS at 37°C and FRET was analyzed using a GE Healthcare INCell 2000 Analyzer.
  • GFP/RFP emission ratio analysis cells were sequentially excited using a FITC filter (490/20) with emission measured using dsRed (605/52) and FITC (525/36) filters, and a polychroic optimized for the FITC/dsRed filter pair (Quad4).
  • CFP/YFP emission ratio analysis cells were sequentially excited using a CFP filter (430/24) with emission measured using YFP (535/30) and CFP (470/24) filters, and a polychroic optimized for the CFP/YFP filter pair (Quad3).
  • HEK293 cells were transfected with 50 ng/well of NKiR with extracellular N-terminal Snap- Tag®. After 48 h, the cell surface NIQR was labeled with SNAP-SurfaceTM 549 photostable fluorescent substrate (New England Biolabs) (1 ⁇ , 30 min, 37°C in DMEM, 0.5% BSA). Cells were washed, recovered in DMEM for 30 min, and stimulated with SP (10 nM, 30 min,
  • HEK293 cells transiently expressing HA-NKiR or HA-CLR/My c-RAMP 1 were loaded with Fura2 ⁇ AM (2 ⁇ ).
  • Fura2 ⁇ AM Fura2 ⁇ AM (2 ⁇ ).
  • Compound 1, L-733,060, Compound 2, CGRP 8"37 , or Compound 4 cells were pre- incubated for 30 min with antagonists, and were then challenged with SP (3 nM, EC 80 ) or CGRP (10 nM, EC 8 o).
  • HEK293 cells transiently transfected with HA-NKiR or HA-NKiR5312 were fixed in PFA (30 min).
  • cells were permeabilized using 0.5% NP-40 in TBS (30 min) after fixation.
  • Cells were incubated in blocking buffer (1% skim milk powder, 0.1M NaHC0 3 , 4 h, RT), and then anti-HA (1 :5,000, Sigma. Overnight, 4°C). Cells were washed and incubated with anti-mouse horseradish peroxidase-conjugated antibody (1 :2,000, 2 h, RT).
  • Cells were washed and stained using the SIGMAFAST OPD substrate (SigmaAldrich). Absorbance at 490 nm was measured using an EnVision plate reader (PerkinElmer Life Sciences). Values were normalized to HEK293 cells transfected with pcDNA3 or to untreated cells.
  • HEK-NKiR cells were plated on poly-D-Lysine coated glass chamber slides or coverslips and cultured for 48 h.
  • To examine uptake of fluorescent SP cells were incubated in HBSS with Alexa568-SP (100 nM, 20 min, 4°C), washed, incubated for 30 min at 37°C, and fixed in 4% paraformaldehyde, 100 mM PBS pH 7.4 (PFA, 20 min, 4°C). Cells were examined by confocal microscopy.
  • NKiR and Go q trafficking cells were incubated in HBSS with SP (100 nM, 15 min) or vehicle and fixed.
  • Cells were blocked in PBS, 0.2% saponin, 3% normal goat serum (1 h, RT). Cells were incubated in primary antibodies: rat anti-HA (1 : 1,000; Roche), rabbit anti-Ga q (1 :2,000, C-19; Santa Cruz Biotechnology), mouse anti-EEAl (1 : 100, 610457; BD Biosciences) (overnight, 4°C). Cells were washed and incubated with donkey anti-rat Alexa488 (1 :500), donkey anti- rabbit Alexa568 (1 : 1,000), and donkey anti-mouse Alexa647 (1 : 1,000) (Life Technologies or Jackson ImmunoResearch) (1 h, RT). Cells were examined by super-resolution microscopy.
  • HEK293 cells were plated on poly-D-lysine-coated glass coverslips. Cells were infected with CellLight® Rab5a-RFP (Life Technologies) or were transfected with rat NKiR-GFP. After 24 h, cells were equilibrated in HBSS, imaged at 37°C by confocal microscopy, and incubated with Cy5-cholestanol or Cy5-ethyl ester (1.5 ⁇ ). Cells expressing NKiR-GFP were incubated with SP (10 nM).
  • Rats Males, 3-8 weeks
  • mice C57BL/6, males, 6-10 weeks
  • Rats Males, 6-10 weeks
  • Rats were maintained in a temperature-controlled environment with a 12 h light/dark cycle and free access to food and water. Animals were killed by anesthetic overdose and thoracotomy.
  • Parasagittal slices (340-400 ⁇ ) were prepared using a vibratome from the lumbar region of the rat spinal cord in ice-cold sucrose-based artificial CSF (sACSF) (mM: 100 sucrose, 63 NaCl, 2.5 KC1, 1.2 NaH 2 P0 4 , 1.2 MgCl 2 , 25 glucose, 25 NaHC0 3 ; 95% 0 2 /5% C0 2 ).
  • sACSF sucrose-based artificial CSF
  • NMDG N-Methyl-D-Glucamine
  • rACSF N-Methyl-D-Glucamine-based recovery ACSF
  • mM 93 NMDG, 93 HC1, 2.5 KC1, 1.2 NaH 2 P0 4 , 30 NaHC0 3 , 20 HEPES, 25 glucose, 5 Na ascorbate, 2 thiourea, 3 Na pyruvate, 10 MgS0 4, 0.5 CaCl 2 ; 95% 0 2 /5% C0 2 , 15 min, 34°C).
  • rat spinal cord Slices of rat spinal cord were transferred to a recording chamber and superfused with normal ACSF (2 ml. min "1 , 36°C). Dodt-contrast optics were used to identify large (capacitance > 20 pF), putative NKiR-positive neurons in lamina I based on their position, size and fusiform shape with dendrites that were restricted to lamina I.
  • Patch electrodes contained KMES-based internal solution (mM: 105 KMES, 20 NaCl, 5 HEPES, 10 BAPTA, 1.5 MgCl 2 , 4 MgATP, 0.4 NaGTP, 0.1% biocytin; 285-295 mosmol.l "1 ) to facilitate subsequent recordings of action potential properties in whole-cell configuration.
  • CNQX (6-cyano-7-nitroqidnoxaline-2,3-dione) (10 ⁇ ; AMPA/kainate receptor antagonist), picrotoxin (100 ⁇ ; GABAA receptor antagonist), strychnine (0.5 ⁇ ; glycine and acetylcholine receptor antagonist), and AP5 ((2A 5 )-amino-5-phosphonovalenc acid; (2/?)-amino-5-phosphonopentanoate) (100 ⁇ ; NMDA receptor antagonist) to minimize presynaptic influences on action potential properties.
  • Slices were challenged by superfusion with SP or CGRP (1 ⁇ ) for 2 min.
  • CGRP stimulated slices were challenged with SP at the end of the experiment to confirm co-expression of CGRP and SP receptors. Recordings were sampled at 10 kHz and filtered with a high pass filter at 1 Hz and firing rate was measured in two-minute interval bins. At the end of each cell-attached recording, whole-cell recordings were made in current-clamp mode to confirm retention of normal action potential firing. Data were only included in the analysis if cells had action potential amplitudes that were >50 mV above threshold to ensure viable neurons were included. Cells were filled with biocytin and sections were processed to confirm NKiR expression by immunofluorescence. The firing rate for each cell was normalized to the response at the 2 min time point, which was not significantly different between groups.
  • the firing time was determined as the duration of the response to last action potential.
  • whole-cell configuration recordings were made under control conditions or after exposure to Dy4 or Dy4 inact; a bipolar stimulating electrode was placed at the dorsal root entry zone, and electrically- evoked excitatory postsynaptic currents recorded as described (Imlach, W. L., et al., Molecular Pharmacology, 2015, 88, 460-428).
  • spinal cord slices 400 ⁇
  • Tissues were then superfused with capsaicin (0.3 ⁇ , 10 min) in the presence of inhibitors or controls.
  • Superfusate (10 min collections, 4 ml) was collected before, during and after capsaicin stimulation, and analyzed for SP-IR and CGRP-IR (Bertin Pharma).
  • Endocytic inhibitors did not interfere with immunoassays. Detection limits of the assays were 2 pg.ml "1 for SP-IR and 5 pg.ml "1 for CGRP-IR. Results are expressed as fmol.g _1 .20 min "1 .
  • NKiR endocytosis in rat spinal neurons NKiR endocytosis in rat spinal neurons.
  • Dy4 inact PS2, PS2 inact (all 50 ⁇ ), or vehicle (1% DMSO/saline) was injected intrathecally (10 ⁇ , L3/L4) into conscious rats. After 30 min, rats were sedated (5% isoflurane) and capsaicin (12.5 ⁇ g) or vehicle (20% ethanol, 10% Tween 80, 70% saline) was injected subcutaneously into the plantar surface of one hindpaw (25 ⁇ ). After 10 min, rats were transcardially perfused with PBS and then 4% PFA. The spinal cord was removed, immersion fixed in PFA (2 h, 4°C), and cryoprotected (30% sucrose, PBS, 24 h, 4°C).
  • the spinal cord (T12 to L4) was embedded in OCT and 30 ⁇ serial coronal sections were cut into 48-well plates containing PBS. Free-floating sections were blocked in PBS containing 10% normal horse serum (1 h, RT). Sections were incubated with mouse anti-NeuN (1:20,000; AbCam) and either rabbit anti-NKiR (1:5,000, #94168) or rabbit anti-pERK (1 :200; Cell Signaling Technology) in PBS containing 3% normal horse serum (48 h, 4°C). Sections were washed (4 x 20 min in PBS) and incubated with donkey anti-rabbit Alexa488 (1:8,000) and donkey anti-mouse Alexa568 (1:2,000 (Life Technologies) (1 h, RT).
  • mice Sections were washed, incubated with DAPI (10 ⁇ g/ml, 5 min), and mounted in Vectashield (Vector Laboratories). Mechanical hyperalgesia, nocifensive behavior and NKjR endocytosis in spinal neurons in mice.
  • mice were acclimatized to the experimental apparatus and environment for 1-2 h on 2 successive days before experiments. Mechanical hyperalgesia was assessed by paw withdrawal to stimulation of the plantar surface of the hind-paw with graded von Frey filaments. On the day before the study, von Frey scores were measured in triplicate to establish a baseline for each animal. To assess edema of the paw, hindpaw thickness was measured using digital calipers before and after treatments. For intraplantar injections, mice were sedated (5% isofiurane).
  • Capsaicin (5 ⁇ g), Complete Freund's Adjuvant (CFA, 2 mg/ml), or vehicle (capsaicin, 20% ethanol, 10% Tween 80, 70% saline; CFA, saline) was injected subcutaneously into the plantar surface of the left hindpaw (10 ⁇ ).
  • von Frey scores left and right paws
  • paw thickness left paw
  • Results are expressed as percent pre- injected values. For assessment of nocifensive behavior, mice were sedated and formalin (4%, 10 ⁇ ) was injected subcutaneously into the plantar surface of the left hindpaw.
  • mice were placed in a Perspex container and nocifensive behavior (flinching, licking, biting of the injected paw) was recorded for 60 min. The total number of nocifensive events was subdivided into acute (I, 0-10 min) and tonic (II, 10-60 min) phases. At the end of experiments, mice were transcardially perfused with PBS and PFA, and the spinal cord was removed and processed to localize the NKiR by immunofluorescence, as described for rats. Investigators were unaware of test agents.
  • Intrathecal injections (5 ⁇ , L3/L4) were made into conscious mice.
  • Dy4 inact, PS2, PS2 inact (all 50 ⁇ ), SR-140333 (15 ⁇ ), SM-19712 (8 mM), U0126 (100 ⁇ ), or vehicle (1% DMSO/saline) was injected intrathecally 30 min before intra-plantar injection of capsaicin or formalin, or 36 h after CFA.
  • Spantide 50 ⁇
  • Compound 1 50 ⁇
  • L- 733,060 100 nM
  • Compound 2 100 nM
  • CGRP 8"37 10 ⁇
  • Compound 4 10 ⁇
  • olcegepant 10 ⁇
  • Cy5-cholestanol 10 ⁇ was injected intrathecally 3 h before or 30 min after intra-plantar injection of capsaicin, 3 h before formalin, or 36 h after CFA.
  • siRNA targeting mouse dynamin-1 (5' (S-S) UAA GUG UCA AUC UGG UCU C dTdT 3') or control siRNA (5' (S-S) CGU ACG CGG AAU ACU UCG AUU dTdT), or siRNA targeting mouse ⁇ -arrl (sense 5' AGC CUU CUG CGC GGA GAA U dTdT 3 1 , antisense 5' dTdT U CGG AAG ACG CGC CUC UUA 5') plus mouse -arr2 (sense: 5' CCU ACA GGG UCA AGG UGA A dT dT 3', antisense: 5' UUC ACC UUG ACC CUG UAG G dT dT 3') or control siRNA (sense: 5' AAG GCC AGA CGC GAA UUA U dT dT, 3
  • Liposome solution cationic lipid 2- ⁇ 3-[bis-(3-amino-propyl)- amino]-propylamino ⁇ -N-ditetradecylcarbamoylmethyl-acetamide (DMAPAP) and L-a- dioleoyl phosphatidylethanolamine (DOPE) (2.5 ⁇ of 200 ⁇ ) was added to siRN A/adjuvant, vortexed for 1 min, and incubated (30 min, RT). The siRNA lipoplexes were administered to mice by intrathecal injection (L1-L4, 5 ⁇ ). After behavioral testing (24-48 h), the spinal cord (L1-L4) was collected for analysis of dynamin-1 expression by Western blotting and ⁇ -arrestin-l and -arrestin-2 expression by q-PCR.
  • DMAPAP cationic lipid 2- ⁇ 3-[bis-(3-amino-propyl)- amino]-propylamino ⁇ -N-ditetradecylcar
  • HEK293 cells were lysed in 150 ⁇ of RIP A buffer containing HALT protease and phosphatase inhibitors (Thermo Scientific). Samples were sonicated on ice, centrifuged and supernatant (20 ⁇ g protein) was fractionated by 10 % SDS-PAGE and transferred to PVDF membranes. Membranes were blocked (1 h, RT) in Odyssey blocking buffer (LI-COR Biosciences), and incubated with sheep anti-dynamin-1 54 (1 : 1,000) or rabbit anti-clathrin (1 ⁇ g/ml, Abeam) antibodies in PBS, 0.2% Tween-20, 50% Odyssey blocking buffer (16 h, 4°C).
  • Odyssey blocking buffer LI-COR Biosciences
  • Membranes were washed and incubated with donkey anti- goat 680 or goat anti-rabbit 680 (1 : 10,000; LI-COR Biosciences) (1 h, RT). Membranes were washed and imaged with the LI-COR Odyssey infrared scanner. Membranes were stripped and re-probed with rabbit anti- -actin (1 : 1,000; Cell Signaling Technology, 16 h, 4°C), washed, incubated with goat anti-rabbit 800 (1 : 10,000, 1 h at RT, LI-COR), and re- imaged. Signals were quantified using ImageJ (NIH). Spinal cord.
  • the dorsal half of the spinal cord was placed in 100 ⁇ of ice cold RIP A buffer containing HALT protease and phosphatase inhibitors (Thermo Scientific). Tissues were homogenized, centrifuged, and supernatant (20 ⁇ g protein) was separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were processed to detect dynamin-1 and ⁇ -actin as described. q-PCR. Mouse lumbar spinal cord (L1-L4) was placed in RNAIater (Qiagen) and total RNA was isolated using RNeasy RNA Isolation kit (Qiagen). Total RNA (500 ng) was reverse- transcribed using SuperscriptTM III cDNA Synthesis Kit (Invitrogen).
  • the relative abundance (R) of each transcript was estimated according to the AC t method using the following formula: 2 ACT .
  • C t is the mean critical threshold at which the increase in fluorescence is the exponential. Assuming efficiency of PCR reaction was 100%, it corresponds to a 2-fold increase in amplicon amount with each cycle of PCR. With this assumption, 2 was used to calculate relative transcript abundance. These values were normalized to an average of housekeeping genes ( ⁇ -actin and GAPHD).
  • mice were acclimatized by 3 trials on 2 successive days before experiments. Mice were trained to remain on the rotarod for three consecutive periods. On the experimental day, three baseline time trials (cut-off 120 s) were recorded. Dy4, PS2, inactive analogues (50 ⁇ ), or vehicle was injected intrathecally (5 ⁇ , L3/L4). After 30 nun, mice were placed on the rotarod with accelerating velocity for up to 120 s. Times were recorded in 3 successive trials, and latency time to fall was determined at 30, 90 and 120 mi a
  • Z stacks were collected of NKiR-positive neurons in lamina I of the dorsal horn. Video projections of Z stacks were made using Imaris Software (Bitplane).
  • NKiR endocytosis and pERK expression were quantified using Image!
  • IR fluorescence was determined in >6 lamina I neurons per condition.
  • the ratio of the number of pERK-IR neurons to total Neu-N-positive neurons in lamina I was determined in >6 fields (x40 objective) per condition.
  • spantide or Compound 1 (10 ⁇ g/ml) was incubated in human cerebrospinal fluid (0-4 h, 37°C), and then snap frozen. Proteins were precipitated using ACN.
  • spantide or Compound 1 was injected intrathecally to mice (10 ⁇ , 5 ⁇ , L3/L4). After 3 hours, a spinal cord segment 5 mm on either side the injection site was removed and snap frozen. Tissues were crushed with a glass rod in a mixture of methanol (30 ⁇ ) and EDTA/KF (60 ⁇ , 0.1 M EDTA, 4 g/1 KF), vortexed and centrifuged to remove lipids.
  • Peptides were extracted from the pellet with 0.5% formic acid in 75% ACN/H 2 0 (100 ⁇ ). Samples were analyzed assayed by LC/MS using a Waters Xevo TQ or TQD triple quadrupole mass spectrometer coupled to a Waters UPLC. Peptides were separated by HPLC (Supelco Ascentis Express Peptide ES C18 column, 50 x 2.1 mm, 2.7 ⁇ ) with 0.05% formic acid in H 2 0 and ACN as solvents. Peptides were quantified by comparison to calibration standards (50-50,000 ng/ml).

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